MXPA00007677A - Tumor-associated antigen derivatives from the mage family, and nucleic acid sequences encoding them, used for the preparation of fusion proteins and of compositions for vaccination - Google Patents
Tumor-associated antigen derivatives from the mage family, and nucleic acid sequences encoding them, used for the preparation of fusion proteins and of compositions for vaccinationInfo
- Publication number
- MXPA00007677A MXPA00007677A MXPA/A/2000/007677A MXPA00007677A MXPA00007677A MX PA00007677 A MXPA00007677 A MX PA00007677A MX PA00007677 A MXPA00007677 A MX PA00007677A MX PA00007677 A MXPA00007677 A MX PA00007677A
- Authority
- MX
- Mexico
- Prior art keywords
- mage
- protein
- leu
- glu
- gly
- Prior art date
Links
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Abstract
The present invention relates to novel proteins and to their production, from the MAGE family. In particular, to a MAGE protein fused to an immunological fusion partner, such as Lipoprotein D. Such antigens may be formulated to provide vaccines for the treatment of a range of tumours. Novel methods for purifying MAGE proteins are also provided.
Description
DERIVATIVES OF ANTIGEN. ASSOCIATED WITH TUMOR. OF THE
FAMILY MAGE. AND NUCLEIC ACID SEQUENCES THAT THE
CODIFICAN: USED FOR THE PREPARATION OF PROTEINS OF
FUSION AND COMPOSITIONS FOR VACCINE
The present invention relates to protein derivatives, comprising an antigen associated with tumor, which has utility in cancer vaccine therapy. In particular, the derivatives of the invention include fusion proteins comprising an antigen encoded by the family of MAGE genes (eg, MAGE-3, MAGE-1), linked to a companion by immunological fusion, which provides T-helper epitopes, such as, for example, the lipidated form of protein D of Haemophilus influenzae B; chemically modified MAGE proteins, in which the disulfide bridges of the antigen are reduced and the resulting thiols blocked; and genetically modified MAGE proteins, provided with an affinity tag and / or genetically modified to prevent disulfide bridge formation. Methods for purifying MAGE proteins and for formulating vaccines for treating a variety of cancers including, but not limited to: melanoma, breast, bladder, lung cancer, non-small cell lung cancer (known as NSCLC), head and squamous cell carcinoma, carcinoma of the colon and carcinoma of the esophagus. The antigens encoded by the MAGE gene family are predominantly expressed in melanoma cells (including malignant melanoma) and in some other cancers, including non-small cell lung cancer (NSCLC), squamous cell carcinoma of the head and of neck, bladder transition cell carcinoma and carcinoma of the esophagus; but they are not detectable in normal tissues, except in the testicles and in the placenta (Gaugler, 1994, Weynants, 1994, Patard, 1995). MAGE-3 is expressed in 69% of melanomas (Gaugler, 1994) and can also be detected in 44% of NSCLC (Yoshimatsu, 1988); 48% of squamous cell carcinoma of the head and neck; 34% of the transition cell carcinoma of the bladder; 57% of esophageal carcinoma; 32% of colon cancers and 24% of breast cancers (Van Peí, 1995, Inoue, 1995, Fujie, 1997, Nishimura, 1997). Cancers that express MAGE proteins are known as tumors associated with Mage. The immunogenicity of human melonoma cells has been elegantly demonstrated in experiments using mixed cultures of melanoma cells and autologous lymphocytes. These cultures frequently generate specific cytotoxic T lymphocytes (called CTL), capable of lysis exclusively of autologous melanoma cells, but not autologous fibroblasts or autologous EBV-transformed B lymphocytes (Knuth, 1984, Anichi, 1987). Several of the antigens recognized in autologous melanoma cells, by these CTL clones, are now identified, including those of the MAGE family.
The first antigen that could be defined by its recognition by specific CTLs in autologous malanoma cells is called MZ2-E (Van den Eynde, 1989) and is encoded by the MAGE-1 gene (Van der Bruggen, 1991). The CTL directed against MZ2-E recognize and effect the lysis of MZ2-E positive melanoma cells, autologous patients as well as other patients, provided that these cells have the HLA.A1 allele. The MAGE-1 gene belongs to a family of 12 intimately related genes: MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, MAGE-7, MAGE-8, MAGE-9 , MAGE-10, MAGE-11, MAGE-12, located on the X chromosome and that share with each other 64 to 85% of homology in their coding sequence (De Plaen, 1994). They are sometimes referred to as MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12 (the MAGE A family). Two other protein groups are also part of the MAGE family, although they are more distantly related. These are the MAGE B group and the MAGE C group. The MAGE B family includes MAGE-B1 (also known as MAGE Xp1)., and DAM 10), MAGE-B2 (also known as MAGE Xp2 and DAM 6), MAGE-B3 and MAGE-B4. The MAGE C family currently includes MAGE-C1 and MAGE-C2. In general terms, an MAGE protein can be defined by containing a core sequence signature located towards the C-terminus of the protein (for example, with respect to MAGE-A1, a protein of 309 amino acids, the core signature corresponds to amino acids 195-279). The consensus pattern of the core signature is thus described as follows; where x represents any amino acid; the residuals in lowercase are conserved (allowed conservative variants) and the residues in uppercase are conserved perfectly. Signature of core sequence: LixvL (2x) l (3x) g (2x) apEExiWex1 (2x) m (3-4x) Gxe (3-4x) gxp (2x) 11 t (3x) VqexYLxYxqVPxsxP (2x) and yeFLWGprA (2x) ) Et (3x) kv Conservative substitutions are well known and are generally established as default annotation matrices in computer programs for sequence alignment.
These programs include PAM250 (Dayhoft M. O. and co-authors (1978), A model of evolutionary changes in proteins ("A model of evolutionary changes in proteins"), in Atlas of Protein sequence and structure
(3) M. O. Dayhoft (ed.), 345-352), National Biomedical Research
Foundation, Washington, and Blosum 62 (Steven Henikoft and Jorja G.
Henikoft (1992), Amino acid substitution matricies from protein blocks ("Matrices of amino acid substitution of protein blocks"), Proc. Nati Acad. Sci USA, 89 (Biochemistry): 10915-10919. Generally speaking, substitution within the following groups is conservative; but substitutions between groups are considered not preserved. The groups are: i) aspartate / asparagine / glutamate / glutamine ii) serine / threonine iii) lysine / arginine iv) phenylalanine / tyrosine / tryptophan v) leucine / isoleucine / valine. methionine vi) glycine / alanine. In general, in the context of this invention, an MAGE protein will be approximately 50% identical in this core region with amino acids 195 to 279 of MAGE-A1. Several etítopes have been identified in the MAGE-3 protein.
CTL. One of these epitopes, MAGE-3.A1, is a non-peptitic sequence located between amino acids 168 and 176 of the MAGE-3 protein, which constitutes a specific epitope for CTLs when they are present in association with the MHC HLA.A1 molecule. of class I. Recently, two additional CTL epitopes have been identified in the peptide sequence of the MAGE-3 protein, due to their ability to mount a CTL response in a mixed culture of melanoma cells and autologous lymphocytes. These two epitopes have sufficient binding motifs for the HLA-A2 alleles (Van der Bruggen, 1994) and HLA.B44 (Herman, 1996), respectively. The present invention provides MAGE protein derivatives. These derivatives are suitable for use in therapeutic vaccine formulations that are suitable for the treatment of a variety of tumor types. In one embodiment of the present invention the derivative is a fusion protein comprising an antigen of the MAGE family of proteins linked to a heterologous partner. The proteins may be chemically conjugated, but are preferably expressed as recombinant fusion proteins, which allow production at increased levels in an expression system, as compared to non-molten proteins. Thus, the fusion partner can help provide epitopes of T helper (immunological fusion partner), preferably T helper epitopes recognized by humans, or can help express the protein (expression enhancer) at yields greater than the natural recombinant protein. It is preferred that the fusion partner be both an immunological fusion partner and an expression enhancing partner. In a preferred form of the invention the immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus infierna B (WO 91/18926). It is preferred that the protein D derivative comprises approximately the first third of the protein, in particular, approximately the first 100 to 110 amino acids of the N-terminus. It is preferred that the protein D derivative be lipidated. Preferably the first 109 residues of the lipoprotein D fusion partner are included in the N-terminus to provide the vaccine candidate antigen, additional exogenous T-cell epitopes and to increase the level of expression in E. coli (acting in that way also as an expression enhancer). The lipid tail ensures the optimal presentation of the antigen to the antigen presenting cells. Other fusion partners include the nonstructural protein of the influenza virus NS1 (hemagglutinin). Typically, N-terminal 81 amino acids are used, although different fragments may be used, as long as they include the epitopes of auxiliary T. In another embodiment, the companion of immune fusion is the protein known as LYTA. It is preferred to use the C-terminal portion of the molecule. Lyta is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alaninamide, the LYTA amidase (encoded by the gene lytA. {Gene, 43 (1986), pages 265-272.}., An autolysin that degrades specifically Certain ligatures in the peptidoglycan skeleton The C-terminal domain of the LYTA protein is responsible for the affinity to choline or some choline analogs, such as DEAE, This property has been exploited for the development of C-LYTA from E. coli , which expresses plasmids useful for the expression of fusion proteins Purification of hybrid proteins containing the LYTA fragment at its amino terminus, has been described [Biotechnology, 10 (1992), pages 795-798.] As used herein, a preferred embodiment uses the repeating portion of the Lyta molecule found at the C-terminal end, from residue 178. A particularly preferred form incorporates residues 188-305. The immunological fusion partners noted above They are also advantageous to help expression. In paticular, such fusions are expressed at higher yields than natural recombinant MAGE proteins. The inventors of the present have demonstrated that said constructions, in a clinical setting, are capable of treating a melanoma. In one case, a patient with stage IV melanoma was free of metastases after two doses of lipoprotein D 1/3
MAGE 3 His, without adjuvant. Accordingly, the present invention, in one embodiment, provides fusion proteins comprising an antigen associated with tumor, of the MAGE family, linked to an immunological fusion partner. It is preferred that the immunological fusion partner be protein D or a fragment thereof, most preferably lipoprotein D. MAGE proteins are preferably MAGE-A1 or MAGE-A3. The lipoprotein D part preferably comprises the first third of the lipoprotein D. The proteins of the present invention are preferably expressed in E. coli. In a preferred embodiment, the proteins are expressed with an affinity tag, such as, for example, a histidine tag comprising between 5 and 9, and preferably six, histidine residues. They are advantageous in their aid for purification.
The present invention also provides a nucleic acid encoding the proteins of the present invention. Said sequences can be inserted into a suitable expression vector and used for DNA / RNA vaccine, or expressed in a suitable host.
Microbial vectors expressing the nucleic acid can be used as vaccines. These vectors include, for example, smallpox virus, adenovirus, alpha virus, listeria and monargo. A DNA sequence encoding the proteins of the present invention can be synthesized using common and current techniques for DNA synthesis, such as enzymatic ligation, as described by DM Roberts and co-authors in Biochemistry, 1985, 24, 5090-5098, by chemical synthesis, by enzymatic polymerization in vitro or by polymerase chain reaction (PCR) technology using, for example, a thermally stable polymerase, or by a combination of those techniques.
Enzymatic DNA polymerization can be performed in vitro using a DNA polymerase, such as DNA polymerase I
(Klenow fragment) in an appropriate regulator containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP, as required, at a temperature of 10 to 37 ° C, generally in a volume of 50 μl or less. Enzymatic ligation of DNA fragments can be carried out using a DNA ligase, such as T4-DNA ligase, in an appropriate regulator, such as 0.05M Tris (pH 7.4), 0.01M MgCl2, 0.01M dithiothreitol, 1 mM of spermidine, 1 mM of ATP and 0.1 mg / ml of bovine serum albumin, at a temperature of 4 ° C to the environment, in general, in a volume of 50 ml or less. The chemical synthesis of the DNA polymer or its fragments can be carried out by conventional chemistry of phosphotriester, phosphite or phosphoramidite, using solid phase techniques, such as those described in Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual (ed. Gassen and A. Lang), Verlag Chemice, Weinheim (1982), or in other scientific publications, for example, MJ Gait, HWD Matthes, M. Singh, BS Sproat and RC Titmas, Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M. D. Metteucci and M. H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S. P. Adams and coauthors, Journal of the American Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J. McMannus and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and HWD Matthes and co-authors, EMBO Journal, 1984, 3, 801. The process of the invention can be carried out by conventional recombinant techniques, such as those described in Maniatis and co-authors, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor, 1982-1989. In particular, the process comprises the steps of: i) preparing a replicable or integrating expression vector capable of expressing, in a host cell, a DNA polymer comprising a nucleotide sequence encoding the protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector; iii) culturing the transformed host cell under conditions that allow the expression of the DNA polymer, to produce the protein; and iv) recover the protein. The term "transform" 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, for example, conventional techniques such as those described in Genetic Engineering, Eds. S. M. Kingsman and A. J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term
"transformed" or "transformant" will be applied subsequently to the resulting host cell that contains and expresses the foreign gene in which there is interest. The expression vectors are novel and also form part of the invention. Replicable expression vectors can be prepared according to the invention, by dividing a vector compatible with the host cell to provide a linear segment of DNA having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with the linear segment, they encode the desired product, such as the DNA polymer encoding the protein of the invention or its derivative, under ligation conditions.
Thus, the DNA polymer can be preformed or formed during construction of the vector, as desired. The selection of the vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic but, preferably, it will be E. coli or CHO cells. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses.
The preparation of the replicable expression vector can be carried out in a conventional manner, with the appropriate enzymes for restriction, polymerization and ligation of the DNA, by the methods described, for example, in Maniatis and co-authors, cited above. The recombinant host cell is prepared, according to the invention, by transforming a host cell with a replicable expression vector of the invention, under transformation conditions. Suitable transformation conditions are conventional and are described, for example, in Maniatis and co-authors, cited above, or in DNA Cloning, Volume II, DM Glover ed., IRL Press Ltd., 1985. The selection of the transformation conditions is determined by the host cell. Thus, a bacterial host, such as E. coli, can be treated with a CaCl 2 solution (Cohen and co-authors, Proc. Nat. Acad. Sci., 1973, 69, 2110), or with a solution comprising a mixture of RbCI , MnCl2, potassium acetate and glycerol, and then with 3- [N-morpholino] propanesulfonic acid, RbCI and glycerol. The mammalian cells of the culture can be transformed by calcium coprecipitation of the vector DNA in the cells.
The invention also extends to a host cell transformed with a replicable expression vector of the invention.
The culture of the transformed host cell is conventionally carried out under conditions that allow the expression of the DNA polymer, as described, for example, in Maniatis and co-authors, and in DNA Cloning, cited above. Thus, nutrients are preferably supplied to the cell and cultured at a temperature below 50 ° C. The product is recovered by conventional methods, according to the host cell and according to the location of the expression product (intracellular or secreted into the culture medium or the periplasm of the cell). Thus, when the host cell is bacterial, such as E. coli, for example, it can be subjected to physical, chemical or enzymatic lysis; and the protein product of the resulting lysate can be isolated. When the host cell is mammalian, the product can be isolated in general from the nutrient medium or cell-free extracts. Conventional protein isolation techniques include: selective precipitation, adsorption chromatography and affinity chromatography, which includes an affinity column for the monoclonal antibody.
The proteins of the present invention are provided either soluble in liquid form or in lyophilized form. It is expected, in general, that each dose for humans comprises from 1 to 1,000 μg of protein and, preferably, from 30 to 300 μg. The present invention also provides a pharmaceutical composition comprising a protein of the present invention, in a pharmaceutically acceptable excipient. A preferred vaccine composition comprises at least lipoprotein D-MAGE-3.
Said vaccine may optionally contain one or more additional antigens, associated with tumor. For example, other members that belong to the MAGE and GAGE families. Suitable tumor associated antigens, OGtros, include MAGE-1, GAGE-1 or tyrosinase proteins. The preparation of the vaccine is described, in general, in Vaccine Design (The subunit and adjuvant approach (eds. Powell M. F. and Newman M. J), (1995), Plenum Press, New York). Encapsulation within liposomes is described by Fullerton, US Pat. No. 4,235,877. Adjuvants are preferably provided for the proteins of the present invention in the vaccine formulation of the invention. The adjuvants include an aluminum salt, such as aluminum hydroxide gel (alum) or aluminum phosphate; but it can also be a calcium, iron or zinc salt, or i can be an insoluble suspension of acylated tyrosine or acylated sugars, cationic or anionically derived polysaccharides, or polyphosphazenes. Other known adjuvants include oligonucleotides containing CpG. The oligonucleotides are characterized in that the CpG dinucleotide is not methylated. These oligonucleotides are well known and are described, for example, in WO 96/02555. In the formulation of the invention it is preferred that the adjuvant composition induces an immunological response of preferably type TH1. Suitable adjuvant systems include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid (3D-MPL), together with an aluminum salt. CpG oligonucleotides also induce, preferably, a TH1 response. An increased system involves the combination of a monophosphoryl lipid A and a saponin derivative, in particular the combination of QS21 and 3D-MPL, as described in WO
94/00153, or a less reactogenic composition, in which QS21 is inactivated with cholesterol, as described in WO 96/33739. A particularly potent adjuvant formulation, which involves QS21, 3D-MPL and tocopherol in an oil-in-water emulsion, is described in WO 95/17210, and is a preferred formulation.
Accordingly, in one embodiment of the present invention there is provided a vaccine comprising a protein of the present invention, more preferably, a lipoprotein D (or a derivative thereof) - MAGE-3, with the monophosphoryl lipid A, or a derivative thereof. He, as an adjuvant. It is preferred that the vaccine further comprises a saponin, more preferably, QS21. Preferably the additional formulation comprises an oil in water emulsion and tocopherol. 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. In one aspect of the invention a process for purifying a MAGE protein produced recombinantly is provided. The process comprises solubilizing the protein, for example in a strong chaotropic agent (such as, for example, urea, guanidium hydrochloride), or in a zwitterionic detergent (e.g., Empigen BB-n-dodecyl-N, N- dimethylglycine); reduce intramolecular and intermolecular disulfide ligatures; block the resulting thiols to prevent oxidant reattachment, and subject the protein to one or more steps of chromatography. It is preferred that the blocking agent be an alkylating agent. Blocking agents of this type include, but are not limited to, alpha-halo acids or alpha-haloamides. For example, iodoacetic acid and iodoacetamide, which result in carboxymethylation or carboxyamidation (carbamidomethylation) of the protein. Other blocking agents can be used and are described in the literature (see, for example, The Proteins, Volume II, eds H. Neurath, RL Hill and CL Boeder, Academic Press, 1976, or Chemical Reagents for Protein Modification, volume I , eds, RL Lundblad and CM Noyes, CRC Press, 1985). Typical examples of these other blocking agents include: N-ethylmaleimide, chloroacetyl phosphate, O-methylisourea and acrylonitrile. The use of the blocking agent is advantageous, since it prevents aggregation of the product and guarantees stability for downstream purification. In one embodiment of the invention, the blocked agents are selected to induce a stable and irreversible covalent derivative (eg, alpha-halo acids or alpha-haloamides). However, other blocking agents can be selected, so that, after purification, the blocking agent can be removed to release the non-derived protein. MAGE proteins that have free derived thiol residues are new and form an aspect of the present invention. In particular, carboxyamidated or carboxymethylated derivatives are preferred embodiments of the invention. In a preferred embodiment of the invention, the proteins of the present invention are provided with an affinity tag, such as CLYTA or a polyhistidine tail. In these cases, the protein, after the blocking step, is preferably subjected to affinity chromatography. For proteins that have a polyhistidine tail, affinity chromatography with immobilized metal ion (IMAC) can be carried out. The metal ion can be any suitable ion, for example: zinc, nickel, iron, magnesium or copper; but preferably it is zinc or nickel. It is preferred that the IMAC regulator contains a hybrid ion detergent, such as Empigen BB (hereinafter referred to as Empigen), as this will result in lower levels of endotoxin in the final product. If the protein is produced with a Clyta part, the protein can be purified by exploiting its affinity for choline or choline analogs, such as DEAE. In one embodiment of the invention, a polyhistidine tail and a Clyta part are provided in the proteins. These can be purified in a simple, two-step affinity chromatographic purification program. The invention will be further described by reference to the following examples.
EXAMPLE I
PREPARATION OF THE RECOMBINANT E. coli STRAIN. WHAT
EXPRESSES THE LIPOPROTEIN FUSION PROTEIN D-MAGE-3-His (LPD 1/3-MAGE-3-HIS OR LPD MAGE-3-His)
1. - The expression system in E. coli.
For the production of lipoprotein D, the DNA encoding protein D has been cloned into the expression vector pMG 81. This plasmid uses signals from lambda phage DNA to drive the transcription and translation of inserted foreign genes. The vector contains the PL PL lambda promoter, the OL operator and two utilization sites (NutL and NutR) to alleviate the effects of polarity by transcription when the N protein is provided (Gross et al., 1985, Mol. &Cell. Biol., 5: 1015). Vectors containing the PL promoter are introduced into a lysogenic host E. coli to stabilize the plasmid DNA. Lysogenic host strains contain lambda phage DNA lacking replication (reproduction), integrated into the genome (Shatzman and co-authors, 1983; In Experimental Manipulation of Gene Expression, Inouya (ed), pages 1-14), Academic Press, NY ). The phage lambda DNA directs the synthesis of the repressor protein cl that binds to the OL repressor of the vector and prevents the binding of the RNA polymerase to the PL promoter and, thus, the transcription of the inserted gene. The gene of expression strain AR58 contains a temperature-sensitive mutation, so transcription directed to PL can be regulated by changes in temperature, that is, an increase in culture temperature inactivates the repressor and the synthesis is initiated of the foreign protein. This system allows the controlled synthesis of foreign proteins, especially those that could be toxic to the cell (Shimataka and Rosenberg, 1981, Nature, 292: 128).
2. - The AR58 strain of E. coli
The strain of lysosomal E. coli AR58, used for the production of the protein LPD-MAGE-3-His is a derivative of the strain N99 of NIH
E. coli K12 (F-su-galK2-lacZ-thr-). It contains a lysogenic, defective lambda phage (galE :: TN10, 1 Kil-cl857 DH1). The Kil phenotype prevents the cancellation of the macromolecular synthesis of the host.
The cl857 mutation confers on the repressor cl a temperature-sensitive lesion. The omission DH1 removes the right operon from phage lambda and the. host sites bio, uvr3 and ch1A. The strain was generated
AR58 by transduction of N99 with a lambda P phage material previously developed in a SA500 derivative (galE :: TN10, 1 Kil-cl857 DH1). The introduction of the defective lysogen in N99 with tetracycline was selected, by virtue of the presence of a TN10 transposon encoding the tetracycline resistance in the adjacent ga1E gene. N99 and SA500 are strains of E coli K12, derived from Dr. Martin Rosenberg's laboratory, at the National Institute of Health.
3. - Construction of the vector designed to express the protein r e com bina nte LPD - MA GE-3 -His
The purpose was to express MAGE-3 as a fusion protein, using the N-terminal third of the lipidated D protein as a fusion partner, connected at the N-terminus of MAGE-3 and a sequence of several histidine residues (His-tail) placed at its C-terminus. Protein D is a lipoprotein (a 42 kDa protein that binds to immunoglobulin D, exposed on the surface of the Gram-negative bacterium Haemophilus influenzae). The protein is synthesized as a precursor with a signal sequence of 18 amino acid residues, which contains a consensual sequence for bacterial lipoprotein (WO 91/18926). When the signal sequence of a lipoprotein is processed during secretion, Cys (at position 19 in the precursor molecule) becomes the amino-terminal residue, and concomitantly is modified by covalent attachment of the ester-linked amino acids and linked to amide. The fatty acids bonded to the amino-terminal cysteine residue then function as membrane anchors. The plasmid expressing the fusion protein was designed to express a precursor protein containing the signal sequence of 18 amino acids and the first 109 residues of the processed protein D; two unrelated amino acids (Met and Asp), amino acid residues 2 to 314 of MAGE-3, two Gly residues that function as a hinge region to expose the subsequent seven His residues. The recombinant strain thus produces the processed, lipidated His-tail fusion protein of 432 amino acid residues long (see Figure 1) with the amino acid sequence described in ID No. 1, and the coding sequence is described in ID No. 2
4. - Cloning strategy for the generation of the fusion protein LPD - MAGE-3-His (vector PRIT144771
A plasmid cDNA was used (from Dr. Thierry Boon of the Ludwig Institute, which contained the coding sequence for the MAGE-3 gene (Gaugler B and co-authors, 1994), and the vector PRIT 14586, which contained the N-terminal portion of the coding sequence Lipo-D-1/3 (prepared as indicated in figure 2) The cloning strategy included the following steps (figure 3): a) .- Amplification by PCR of the sequences present in the
Plasmid cDNA MAGE-3, using the sense of the oligonucleotide: 5 'ge gcc atg gat ctg gaa cag cgt agt cag falls tgc aag cct; and the opposite direction to the oligonucleotide: 5 'gcg tet aga tta atg gtg atg gtg atg gtg atg acc gcc etc ttc ecc etc tet caá); this amplification leads to the following modifications at the N-terminus: change of the first five codons to the use of the E. coli codon; Codon Pro replacement by an Asp codon at position 1; installation of a Ncol site at the 5 'end and, finally, the addition of two codons 2 Gly and codon 7 His, followed by a Xbal site at the C end. b) Cloning in the TA cloning vector of invitrogen , of the above amplified fragment and preparation of the intermediate vector pRIT14647. c) .- Extirpation of the Ncol Xbal fragment of the plasmid pRIT14647 and cloning in the vector pRIT 14586. d) .- Transformation of the host strain AR58. e) .- Selection and characterization of the transformants of the E. coli strain, which contain the plasmid pRIT 14477, which express the LPD-MAGE-3-His fusion protein.
EXAMPLE II
PREPARATION OF THE ANTIGEN LPD1 / 3 - MAGE-3-HIS
1. - Development and induction of the bacterial strain - Expression of LPD1 / 3 - MAGE-3-His
AR58 cells transformed with the plasmid pRIT14477 were developed in two liter flasks, each containing 400 ml of LY12 medium, supplemented with yeast extract (5.4 g / liter) and kanamycin sulfate (50 mg / liter). After incubation on a shaking table at 30 ° C for 8 ± 1 hours, a small sample was taken from each flask for examination under a microscope. The contents of the two flasks were combined to give the inoculum for the 20 liter fermenter. The inoculum (approximately 800 ml) was added to a fermentor of 20 liters (of total volume), previously sterilized, containing 7 liters of medium, supplemented with 50 mg / liter of kanamycin sulfate. The pH was adjusted to 6.8 and maintained at that value by the periodic addition of ammonium hydroxide (25% volume / volume), and the temperature was adjusted to 30 ° C and maintained at that value. The aeration rate was adjusted to 12 liters of air / minute and maintained at that value; and the dissolved oxygen tension was maintained at 50% saturation by the feedback control of the stirring speed. The excess pressure in the fermenter was maintained at 500 g / cm2 (0.5 baria). Cultivation was carried out by intermittent feeding, by the controlled addition of a carbon feed solution. The feed solution was added at an initial rate of 0.04 ml / minute and increased exponentially during the first 42 hours, to maintain a development rate of 0.1 ir1. After 42 hours, the temperature of the fermenter rapidly increased to 39 ° C, and the feed rate was maintained constant at 0.005 ml / g DCW / minute during the induction phase, for another 22-23 hours, during which time the Intracellular expression of LPD - MAGE-3-His reached the maximum level.
Aliquots of 15 ml of the broth were taken at regular intervals during all the development / induction phases, and at the end of the fermentation, to follow the kinetics of the microbial development and the intracellular expression of the product and, additionally, to provide samples for the tests of microbe / purity identification. At the end of the fermentation, the optical density of the culture was between 80 and 120 (which corresponds to a cellular concentration of between 48 and 72 g of DCW / liter), and the total liquid volume was approximately 12 liters. The culture was rapidly cooled to between 6 and 10 ° C and the ECK32 cells were separated from the culture broth by centrifugation at 5000 x g, at 4 ° C, for thirty minutes. The concentrated ECK32 cells were quickly stored in plastic bags and immediately frozen at -80 ° C.
2. - Protein extraction
The concentrated and frozen ECK32 cells were thawed at 4 ° C before being resuspended in the cell disruption buffer at a final optical density of 60 (corresponding to a cell concentration of approximately 36 g of DCW / liter). ).
The cells were broken by passing them twice through a high pressure homogenizer (1,000 bars). The suspension of broken cells was centrifuged (x 10,000g at 4 ° C for 30 minutes) and the pellet fraction was washed twice with Triton X100 (1% w / v) + EDTA (1mM), and then washed with phosphate buffered saline (PBS) + Tween 20 (0.1% volume / volume) and finally washed with PBS. Between each washing step, the suspension was centrifuged at x 10,000 g for thirty minutes at 4 ° C, the supernatant was discarded and the pellet fraction was retained.
EXAMPLE III
CHARACTERIZATION OF THE PROTEIN LIPO D - MAGE-3
Purification
LPD-MAGE-3-His was purified from the cell homogenate, using a sequence of steps described below: a) Solubilization of the washed pellet fraction, from cell disruption; b) chemical reduction of the disulfide bonds within the protein and between the proteins, and then blocking the thiol groups, to prevent oxidant re-coupling; c) microfiltration of the reaction mixture to remove particles and reduce endotoxins.
d) capture and primary purification of LPD-MAGE-3-His by exploitation of the affinity interaction between the polyhistidine tail and the Chelating Sepharose, loaded with zinc; e) elimination of contaminating proteins, by means of anion exchange chromatography. The purified LPD-MAGE-3-His was subjected to several polishing steps: f) Regulation change / urea removal, by means of size exclusion chromatography, using Superdex 75. g) Filtration in process. h) Change of regulator / desalination, by means of chromatography by size exclusion, using Sephadex G25. Each of these steps is described in more detail below: 1.1) .- Solubilization of the cell homogenate pellet:
The pellet fraction of the final wash step (as described above) was again solubilized, overnight, in 800 ml of a 6M solution of guanidine hydrochloride and 0.1 MN of sodium phosphate (pH 7.0), a 4 ° C. 1.2) .- Reduction and carboxymethylation: The solubilized material (a cloudy, pale yellow suspension) was flooded with argon, to purge any remaining oxygen; and a master solution of 2-mercaptoethanol (14M) was added to give a final concentration of 4.3M (corresponding to 0.44 ml of 2-mercaptoethanol per ml of solution).
The resulting solution was divided and transferred to two glass flasks that were heated to 95 ° C in a water bath. After 15 minutes at 95 ° C the flasks were taken out of the water bath and allowed to cool, after which the contents were gathered in a 5 liter beaker, covered with foil; it was placed on ice and iodoacetamide was added with vigorous mixing, to give a final concentration of 6M (corresponding to 1.11 g of iodoacetamide per ml of solution). The mixture was kept on ice and in the dark for one hour, to ensure complete solubilization of the iodoacetamide, before it was neutralized (maintaining vigorous mixing and continuously monitoring the pH), by the addition of approximately one liter of sodium hydroxide. (5M) to give a final pH of 7.5-7.8. The resulting mixture was kept on ice and in the dark for another 30 minutes, after which the pH was again adjusted to 7.5-7.8. 1.3) .- Microfiltration: The mixture was microfiltered in an Amicon Proflux M12 unit, tangential flow, equipped with a hollow fiber cartridge Minikros (reference number M22M-600-01 N, area, 5,600 cm2, 0.2 μm). The permeate was retained for subsequent chromatographic purification. 1.4) .- Chromatography of metallic chelate (Zn2 +) (IMAC): Metallic chelate chromatography was carried out with
Chelating Sepharose FF (Pharmacia Biotechnology, Catalog No. 18-1103-01). The dimensions of the packed bed were: diameter, 10 cm; cross sectional area, 79 cm2; bed height, 19 cm; packed volume, 1,500 ml. The empty column was sanitized with sodium hydroxide (0.5M), then washed with purified water. The support (supplied in 20% volume / volume of ethanol), with 8 liters of purified water, was washed in a Buchner funnel (vacuum) and charged with zinc, passing at least 15 liters of a 0.1 solution. M of ZnCl2. Excess zinc was removed by washing the support with 10 liters of purified water until the pH of the exit liquid reached the pH of the ZnCl 2 solution (pH 5.0). The support was then equilibrated with 4 liters of a solution containing guanidine hydrochloride (6M) and sodium phosphate (0.1M, pH 7.0). The permeate from the microfiltration, containing LPD-MAGE-3-His, was mixed with the support (intermittent binding) before loading and packing the BPG column with a solution containing guanidine hydrochloride (6M) and sodium phosphate (0.1 M, pH 7.0). The following steps of metal chelate chromatography were carried out at an eluent flow rate of 60 ml / minute. The column was washed first with the solution containing guanidine hydrochloride (6M) and sodium phosphate (0.1M, pH 7.0), then with the solution containing urea (6M) and sodium phosphate (0.1M, pH 7.0), until the eluent of the column reached zero absorbance at OD28o nm (baseline). The semipure fraction of LPD-MAGE-3-His protein was eluted with 2 column volumes of a solution containing urea (6M), sodium phosphate (0.1M, pH 70) and imidazole (0.5M). The conductance of this fraction was approximately 16 mS / cm. 1.5) .- Chromatography of change of anions. Before proceeding with the anion exchange chromatography, the conductance of the LPD-MAGE-3-His semipure protein fraction was reduced to approximately 4 mS / cm, diluting with a solution containing urea (6M) and Tris-HCl (20). mM, pH 8.0). Anion exchange chromatography was carried out using Q-Sepharose FF (Pharmacia Biotechnology, Catalog No. 17-0510-01) in a pap on a BPG 200/500 column (Pharmacia Biotechnology, Catalog No. 18-1103- eleven). The dimensions of the packed bed were: diameter, 10 cm; cross sectional area 314 cm2; bed height 9 cm; packed volume, 2,900 ml. The column was packed (with 20% volume / volume of ethanol) and washed with 9 liters of purified water, at an eluent flow rate of 70 ml / minute. The packed column was sanitized with 3 liters of sodium hydroxide (0.5M), washed with 30 liters of purified water, then equilibrated with six liters of a solution containing urea (6M) and Tris-HCl (20mM, pH 8.0). The diluted, semi-purified MDA-3-His LPD was loaded onto the column and then washed with 9 liters of a solution containing urea (6M), Tris-HCl (20mM, pH 8.0), EDTA (1mM) and Tween (0.1%), until the absorbance (280 nm) of the eluent fell to zero. Another wash step was carried out with six liters of a solution containing urea (6M) and Tris-HCl (20mM, pH 8.0).
The purified LPD-MAGE-3-His was eluted from the column with a solution containing urea (6M), Tris-HCl (20mM, pH 8.0) and NaCl (0.25M). 1.6) .- Chromatography by size exclusion: Both the removal of urea from purified LPD-MAGE-3-His, as well as the change of regulator, was obtained by size exclusion chromatography. This was accomplished using Superdex 75 (Pharmacia Biotechnology, Catalog No. 17-1044-01), packaged on an XK 50/100 column (Pharmacia Biotechnology, Catalog No. 18-8753-01). The dimensions of the packed bed were: diameter, 5 cm; cross-sectional area, 19.6 cm2; bed height 90 cm; packed volume, 1,800 ml. The column was packed in ethanol (20%) and washed with 5 liters of purified water, at an effluent flow rate of 20 ml / minute. The column was sanitized with 2 liters of sodium hydroxide (0.5M), it was washed with 5 liters of purified water, then it was equilibrated with 5 liters of phosphate regulated salt, which contained Tween 80 (0.1% volume / volume). The purified fraction of LPD-MAGE-3-His (maximum 500 ml / desalting operation) was loaded onto the column at an effluent flow rate of 20 ml / minute. Purified LPD-MAGE-3-His, desalted, was eluted from the column with 3 liters of PBS containing Tween 80 (0.1% volume / volume). The fraction containing LPD-MAGE-3-His eluted in the hollow volume of the column.
1. 7) .- Filtration in process: The volume of LPD - MAGE - 3 - His from the chromatography by size exclusion was filtered through a membrane of 0.22 μm, in a laminar flow hood (class 10,000). The filtered volume was frozen at -80 ° C and stored until the desalting step. 1.8) .- Desalination Chromatography: Since the osmolarity of the final volume must be less than 400 mOsM, it was necessary to change the regulator gold step to reduce the salt concentration. This was carried out by means of a desalting chromatographic step using Sephadex G25 (Pharmacia Biotechnology, Catalog No. 17-0033-02) packed in a BPG 100/950 column (Pharmacia Biotechnology, Catáloco No. 18-1103-03) . The dimensions of the packed bed were: diameter, 10 cm; cross sectional area, 78.6 cm2; bed height, 85 cm; packed volume 6,500 ml. The Sephadex G25 was hydrated with 7 liters of purified water and allowed to swell overnight at 4 ° C. The gel was then packed in the column with pure water, at an eluent flow rate of 100 ml / min. The column was sanitized with 6 liters of sodium hydroxide
(0.5M), then equilibrated with 10 liters of a solution containing sodium phosphate (10 mM, pH 6.8), NaCl (20 mM) and Tween 80 (0.1% volume / volume). The purified fraction of LPD-MAGE-3-His (maximum 1,500 ml / desalting step) was loaded onto the column at an eluent flow rate of 100 ml / minute. The desalinated and purified fraction of LPD-MAGE-3-His eluted in the hollow volume of the column was filtered sterile through a 0.22 μm membrane and stored at -80 ° C. The final volume protein was thawed at + 4 ° C, before being divided into aliquots for ampoules and dried by freezing in a lactose excipient (3.2%).
2. - Analysis in which SDS-polyacrylamide. dyed with Coomassie
The purified LPD-MAGE-3-His antigen was analyzed by SDS-PAGE on a 12.5% acrylamide gel, under reducing conditions. The protein loading was 50 μg for staining with Coomassie blue, and 5 μg for staining with silver nitrate. Clinical lot 96K19 and pilot lot 96J22 were analyzed. A main band was visualized, which corresponds to a molecular weight of 60 kDa. Two additional minor bands, approximately 45 kDa and 35 kDa, were also observed.
3. - Western blot analysis
Peptides revealed by SDS-PAGE analysis of the LPD-MAGE-3-His protein were identified by Western blot using mouse monoclonal antibodies. These antibodies were revealed at home, using a preparation purified by the MAGE-3-His protein (this protein does not contain the LPD part of the LPD -MAGE-3-His). Two preparations of monoclonal antibody (Mab 22 and Mab 54) had been selected on the basis that they were suitable for Western blot analysis and used in the identity test for batch emission. Figure 4 shows the band patterns obtained for batches 96K19 and 96J22, after staining with Mabs 32 and 654. Six hundred (600) ng of protein were resolved on a 12.5% SDS-PAGE, transferred to a membrane of nylon, was reacted with Mabs 32 and 54 (6.0 μg / ml) and was revealed with anti-mouse antibodies coupled to peroxidase. The 60 kDa and 30 kDa peptide detected by SDS-PAGE was revealed by both Mabs.
EXAMPLE IV
1. - Preparation of vaccine using the protein LPD - MAGE-3-His
The vaccine used in these experiments is produced from a recombinant DNA, which encodes a 1/3-IMAGE-3-His lipoprotein, expressed in E. coli from the AR58 strain, either with or without adjuvant. As an adjuvant, the formulation comprises a mixture of de-O-acylated monophosphoryl lipid A (3D-MPL) and QS21 in an oil / water emulsion. The SBAS2 adjuvant system has been previously described in WO 95/17210. 3D-MPL: It is an immunostimulant derived from the polysaccharide (LPS) of the Gram-negative bacterium Salmonella minnesota. MPL has been deacylated and lacks a phosphate group in the lipid A portion. This chemical treatment dramatically reduces toxicity, while maintaining immunostimulating properties (Ribi, 1986). Ribi immunochemistry produces and delivers MPL to SB-Biologicals. The experiments carried out in SmithKIine
Beecham Biologicals have shown that 3D-MPL, combined with various vehicles, strongly increases both the humoral type and the TH1 type of cellular immunity. QS21: It is a natural saponin molecule, extracted from the bark of the South American tree Quillaja saponiaria Molina. A purification technique developed to separate the individual saponins from the raw extracts of the bark, allowed the isolation of the particular saponin QS21, which is a triterpene glycoside, which shows to have stronger adjuvant activity and lower toxicity, compared to the predecessor component. QS21 has been shown to activate CTLs restricted with MHC class I, to several subunit Ags, as well as to stimulate the proliferation of Ag-specific lymphocytes (Kensil, 1992). Aquila (formerly Cambridge Biotech Corporation) produces and supplies QS21 to SB-Biologicals.
The experiments carried out in SmithKIine Beecham Biologicals have shown a clear synergistic effect of the combinations of MPL and QS21 in the induction of cellular immune responses of both humoral and TH1 type. The oil-in-water emulsion is composed of an organic phase made of two oils (a tocopherol and squalene) and an aqueous phase of PBS containing Tween 80 as an emulsifier. The emulsion consisted of 5% squalene, 5% tocopherol, 0.4% Tween 80 and had an average particle size of 180 nm, and is known as SB2 (see WO 95/17210). The experiments carried out in SmithKIine Beecham Biologicals have shown that the assembly of this oil-in-water emulsion to 3D-MPL / QS21 (SBAS2) further increases the immunostimulatory properties of the latter, against various subunit antigens.
2. - Preparation of emulsion of SB62 (double concentrate)
Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in PBS. To give 100 ml of double concentrated emulsion, 5 g of DL-alpha-tocopherol and 5 ml of squalene are stirred until vortex is formed, to mix them perfectly. Then 90 ml of the PBS / Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and, finally, is microfluidized using an M110S microfluidizing machine. The resulting oil droplets are approximately 180 nm in size.
3. - Preparation of Lipoprot. D1 / 3 - MAGE-3-His QS21 / 3D MPL. formulation of oil in water (SBAS2)
The adjuvant is formulated as a combination of MPL and QS21, in an oil / water emulsion. This preparation is supplied in 0.7 ml ampoules to be mixed with the lyophilized antigen (the ampoules contain 30 to 300 μg of antigen). The composition of the adjuvant diluent for the lyophilized vaccine is as follows:
The final vaccine is obtained after reconstitution of the lyophilized preparation of LPD-MAGE-3-His, with the adjuvant or with PBS only. Controls of adjuvants without antigen were prepared by replacing the protein with PBS.
4. - VACCINE ANTIGEN: LIPOPROTEIN OF FUSION PROTEIN D1 / 3 - MAGE-3-His
Lipoprotein D is a lipoprotein exposed on the surface of the Gram-negative bacterium Haemophilus influenzae. The inclusion of the first 109 residues of the processed D protein, as a fusion partner, is incorporated to provide T-cell epitopes in the vaccine antigen. In addition to the LPD portion, the protein contains two unrelated amino acids (Met and Asp) , amino acid residues 2 to 314 of Mage-3, two Gly residues that function as a hinge region to expose the seven subsequent His residues.
EXAMPLE V 1.- Immunogenicity of LPD - MAGE-3-His in mice and monkeys
In order to test the antigenicity and immunogenicity of the human MAGE-3 protein, the candidate vaccine was injected into two different strains of mice (C57BL / 6 and Balb / C), which vary in their background alleles and genetic MHC. For both mouse strains the potential motifs of MHC class I and MCH class II peptide were predicted theoretically, for the MAGE part of the LPD-MAGE-3-His fusion protein. a) The immunization protocol: 5 mice of each strain were injected twice at two week intervals in the paw plant, with 5 μg of LPD-MAGE-3-His, formulated or not in SBAS2, at 1/10 of the concentration used in human controls. b) Proliferation analysis: Lymphocytes were prepared by grinding the spleen or popitle lymph nodes of the mice two weeks after the last injection. 2 x 105 cells were placed in triplicate in plates of 96 concavities and the cells were re-stimulated in vitro for 72 hours with different concentrations (1-0.1 μg / ml) of His-Mage-3, as such, or coated on microgranules. latex A specific lymphoproliferative activity was observed for
MAGE-3, increased, with spleen cells (see Figures 5 and 7) and lymph node cells (see Figures and 8) of C57BL / 6 or Balb / C mice, injected with the LPD-MAGE-3 protein His, compared to the lymphoproliferative response of mice that had received the formulation SBAS-2 alone or PBS. In addition, a greater, significant proliferative response was obtained with the lymphocytes of mice immunized with LPD -MAGE-3-His in the SBAS2 adjuvant (see figures 6 and 8). c) .- Conclusion: LPD - MAGE-3-His is immunogenic in mice and this immunogenicity can be increased by the use of the adjuvant formulation SBAS2.
2. - ANTIBODY RESPONSE
a) .- Immunization protocol: Balb / C or C57BL / 6 mice were immunized by two injections in the paw plant, at two weeks interval, with PBS or with SBAS2 or with 5 μG of LPD - MAGE-3 His or with 5 μG of LPD - MAGE-3-His + SBAS2. Three and five animals were used in the control groups and in the test groups, respectively. b) Indirect ELISA Two weeks after the second injection, individual sera were taken and subjected to indirect ELISA. 2 μg / ml of His MAGE-3 was used as applied antigen.
After saturating for one hour at 37 ° C in PBS + 1% newborn calf serum, the sera were serially diluted (starting at 1/1000) in the saturation buffer and incubated overnight at 4 ° C or 90 minutes at 37 ° C. After washing in PBS / 20.01% Tween, biotinylated goat anti-mouse IgG total antisera (1/1000) or goat anti-mouse IgG1, IgG2a, IgG2b (1 / 5,000) antisera were used as second antibodies. Streptavidin coupled to peroxidase was added, and TMB (tetramethylbenzidine peroxide) was used as the substrate. After 10 minutes the reaction was blocked by adding 0.5M sulfuric acid and the O.D. c) .- The results: Figure 9 compares the different groups of mice (N = 5 / group), the middle point title, relative average, of the sera, which consists of the average dilution needed to reach the midpoint of the curves. These results show that, in both strains of mice tested, a weak Ab response is mounted after two injections of LPD-MAGE-3-His alone, but that higher anti-MAGE 3 Ab concentrations are generated when LPD-MAGE- is injected. 3-His in the presence of SBAS2. Thus, only two injections of LPDP-MAGE-3-His + SBAS2 are sufficient, at two weeks interval, to generate the elevated Ab response, observed. The best Ab response observed in Balb / c mice, compared to the response obtained in C57BL / 6 mice, can be explained by the differences in haplotypes or in the antecedents between these two races, even though the Ab title obtained in the C57BL / 6 mice are also higher after injections of LPD-MAGE-3-His + SBAS2 than after injections with LPD-MAGE-3-His alone. The anti-MAGE-3 responses specific for the Ig subclasses can be seen in FIGS. 10 and 11, after the vaccines in the different groups of mice, which gives a comparison of the mean dilution of the sera midpoint. No IgA or IgM was detected in any of the serum samples, even from the mice vaccinated with LPD-MAGE-3-His in the SBAS2 adjuvant. In contrast, the total IgG level was slightly higher in the sera of mice vaccinated with LPD-MAGE-3-His alone, and significantly increased in the sera of the animals injected with LPD-MAGE-3-His in SBAS2. The analysis of the concentrations in the different IgG subclasses shows that a mixed Ab response was induced in the mice, since the levels of all the IgG subclasses tested (IgG 1, IgG2a, IgG2b) were higher in the mice vaccinated with the Ag with adjuvant than in mice injected with Ag or adjuvant alone. The nature of the mixed Ab response after the LipoD-MAGE-3 vaccine in the presence of SBAS2, however, appears to depend on the race of mice, since IgG 1 and IgG2b were predominantly found in the sera of the Balb mice. cy C57BL / 6, respectively.
3. - Immunochemistry of Lipoprotein D 1/3 MAGE-3-His + SBAS2 adjuvant in Rhesus monkeys
Three groups of five Rhesus animals were selected. { Macaca mulatta). RTS, S and gp120 were used as a positive control. Groups: Group 1 right leg: RTS.S / SBAS2 left leg: GP120 / SBAS2 Group 2 right leg: RTS.S / SB26T left leg: GP120 / SB26T Group 3 right leg: LipoD1 / 3 Mage 3 His / SBAS2. The animals were vaccinated on day 0 and the vaccine was boosted on day 28 and 84 and they were bled to determine their antibody response to both MAGE 3 and the protein D component. The vaccines were administered intramuscularly as a bolus injection
(0.5 ml) on the back of the right leg. Small blood samples were taken every 14 days. 3-heparinized blood samples were collected from the femoral vein, allowed to clot for at least one hour and centrifuged at room temperature for 10 minutes at 2,500 rpm. Serum was removed, frozen at -20 ° C and sent for determination of antibody levels by specific Elisa. Microplates of 96 concavities (Maxisorb Nunc) were coated with 5 μg of His Mage-3 or with protein D overnight, at 4 ° C. After saturation for one hour at 37 ° C with 1% PBS NCS, the serial dilution of the rabbit sera was added for one hour and thirty minutes at 37 ° C (beginning at 1/10), after three washes in PBS Tween rabbit biotinylated serum was added (Amersham, ref RPN 1004, lot 88) (1/5000). The plates were washed and streptavidin coupled with peroxidase (1/5000) was added for thirty minutes at 37 ° C. After washing, 50 μl of TMB (BioRad) was added for seven minutes and the reaction was stopped with 0.2M sulfuric acid; the OD at 450 nm was measured. The midpoint dilutions were calculated by SoftmaxPro. Antibody response: Small blood samples were taken every 14 days to follow the kinetics of the antibody response to Mage 3, by ELISA. The results indicate that, after an injection of LPD1 / 3 Mage-3-His + SBAS2 the total Ig-specific titer for Mage-3 was low; a clear booster was seen in 3 out of every 5 animals after a second and third injections of LipoD1 / 3 Mage-3 + adjuvant, in the same monkeys. Those who responded poorly remained negative even after the three injections. Twenty-eight days after II or after lll, antibody titers had returned to basic levels. The subclass of these antibodies that were predominantly IgG and not IgM was determined. The change to IgG suggests that a T helper response was triggered. The antibody response specific for protein D, although weaker, is exactly parallel to the Mage-3 antibody response.
EXAMPLE VI
1. - LPD - MAGE-1-His
Analogously, LPD-MAGE-1-His was prepared. The amino acid and DNA sequences are illustrated in sequences ID No. 3 and 4. The resulting protein was purified analogously to the LPD-MAGE-3-His protein. Briefly, the cell culture was homogenized and treated with 4M guanidine hydrochloride and 0.5M beta-mercaptoethanol, in the presence of 0.5% Empigen detergent. The product was filtered and the permeate was treated with 0.6M of iodoacetamide. The carboxyamide fractions were subjected to IMAC chromatography (Chelate-sepharose FF zinc). The column was first equilibrated and washed with a solution containing 4M of guanidine hydrochloride and sodium phosphate (20 mM, pH 7.5) and 0.5% of Empigen; then the column was washed with a solution containing 4M urea in sodium phosphate (20 mM, pH 7.5), 0.5% Empigen regulator. The protein was eluted in the same buffer, but with an increasing concentration of imidazole (20 mM, 400 mM and 500 mM).
The eluate was diluted with 4M urea. The Q-sepharose column was equilibrated and washed with 4M urea in 20 mM phosphate buffer (pH 7.5) in the presence of 0.5% Empigen. A second wash was carried out in the same regulator, but devoid of the detergent. The protein was eluted in the same regulator but with increasing imidazole (150 mM, 400 mM, 1M). The eluate was ultrafiltered.
EXAMPLE VII
CONSTRUCTION OF THE EXPRESSION PLASMIDE PRIT14426 AND TRANSFORMATION OF THE HEAVY BOW AR58 TO PRODUCE NS1-MAGE-3-HIS.
Protein design: The design of the fusion protein NS1, -MAGE-3-His to be expressed in E. coli is described in figure 12. The primary structure of the resulting protein has the sequence indicated in ID No. 5. The coding sequence (ID No. 6) corresponding to the previous protein design was placed under the control of the? PL promoter in an E. coli expression plasmid.
THE CLONING STRATEGY FOR THE GENERATION OF THE NS-MAGE-3-His FUSION PROTEIN The starting material was a cDNA plasmid, received from the
Dr. Tierry Boon, of the Ludwig Institute, which contained the coding sequence for the MAGE-3 gene and the PMG81 vector, which contained the 81 amino acid coding region of NST (non-structural protein) of influenza. The cloning strategy indicated in figure 13 included the following steps: a) PCR amplification of the sequences present in the cDNA-MAGE-3 plasmid, using the sense of the oligonucleotide: 5 'g gcc atg gat ctg gaa cag cgt agt cag falls tgc aag cct; and the opposite direction of oligonucleotide: 5 'gcg tet aga tta atg gtg atg gtg atg gtg atg acc gcc etc ttc ecc etc tet caá. The amplification leads to the following modifications at the N-terminus: change of the first five codons to the use of the E. coli codon; Codon Pro replacement by an Asp codon at position 1; installation of a Ncol site at the 5 'end and finally addition of codons 2 Gly and codon 7 His, followed by a Xbal site at the C-terminus. b) Cloning into the invitrogen TA cloning vector of the above amplified fragment, and preparation of the intermediate vector pRIT14647. c) Removal of the Ncol Xbal fragment from plasmid pRIT14647 and cloning into the pRIT PMG81 vector. d) Transformation of host strain AR58. e) Selection and characterization of the transformants of the E. coli strain containing the plasmid pRIT14426 (see Figure 14) expressing the NS1-MAGE-3-His fusion protein.
CHARACTERIZATION OF RECOMBINANT NS ^ MAGE-3-His (PRIT14426)
Bacteria were developed in LB medium supplemented with 50 μg / ml kanamycin at 30 ° C. When the culture had reached OD = 0.3 (at 620 nm), induction was obtained by raising the temperature to 42 ° C. After four hours of induction, the cells were harvested, resuspended in PBS and lysed (by disintegration) by pressing three times in the French press. After centrifugation (60 minutes at 100,000 g) the pellet supernatant and the total extract were analyzed by SDS-PAGE. The proteins were visualized on gels stained with Coomassie B 1, where the fusion protein represented approximately 1% of the total of E. coli proteins. The recombinant protein appeared as a single band with an apparent molecular weight of 44.9 K. The fusion protein was identified by Western blot analysis using anti-NS 1 monoclonal.
EXAMPLE VIII
PURIFICATION OF NS1 -MAGE-3-HIS (E. coli) FOR IMMUNIZATION OF RABBIT / MOUSE
Purification scheme: The following purification scheme was used to purify the antigen: Cell lysis + centrifugation
Soil antilubrication + centrifugation
Ni 12 + -NTA agarose
Co Incentration
Prescription with TCA and solubilization in PBS. a) Lysis 23 g of bacterial cells were lysed in 203 ml of a 50 mM regulator of PO 4, pH 7, by Rannie (homogenizer) and then the lysate was centrifuged in a JA 20 rotor of 15,000 rpm for thirty minutes. The supernatant was discarded. b) Solubilization of antigen The 1/3 pellet O / N was again solubilized at 4 ° C in 34 ml of 100 mM P0 -6M GuHCl, pH 7. After centrifugation in a JA rotor at 15,000 rpm for 30 minutes minutes, the pellet was discarded and the supernatant was further purified, by IMAC. c) Affinity chromatography: Ni2 + -NTA aqarose (Qiaqen) Column volume: 15 ml (16 mm x 7.5 cm) Packing regulator: 0.1M of PO4-6M of GuHCI, pH 7. Sample regulator: the same. Washing regulator: 0.1M of PO4-6M of GuHCI, pH 7, 0.1M of PO4-6M of urea, pH 7. Elution: gradient of imidazole (0-250mM) in 0.1M of regulator PO, pH7, supplemented with 6M of urea. Flow rate: 2 ml / min. a) Concentration: The antigen-positive fractions of the eluted IMAC (160 ml) were pooled and concentrated to 5 ml of an Amicon stirred cell on a membrane (Omega type, cut 10,000). The purity in this stage is approximately 70%, when estimated by SDS-PAGE. b) Preparatory electrophoresis (Prep Cell Biorad) 2.4 ml of the concentrated sample was boiled in 0.8 ml of reducing sample buffer and loaded on a 10% acrylamide gel. The antigen was eluted in a Tris-glycine buffer, pH 8.3, supplemented with 4% SDS and the positive fractions were combined with Ns -? - MAGE-3-His. a.- Precipitation with TCA. The antigen was precipitated with TCA and, after centrifugation in a JA 20 rotor at 15,000 rpm for 20 minutes, the supernatant was discarded. The pellet was again solubilized in PBS buffer, pH 7.4. The protein is soluble in PBS; after freezing / thawing it shows no degradation when stored for three hours at 37 ° C and has an apparent molecular weight of approximately 50,000 daltons, when determined by SDS (PAGE, 12.5%).
EXAMPLE IX
PREPARATION OF E. coli CEPA EXPRESSING A FUSION PROTEIN CLYTA-MAGE-1 - COLA His
1. - Construction of the expression plasmid pRIT14613 and transformation of the host strain AR58.
Protein Design: The design of the Clyta-Mage-1-His fusion protein to be expressed in E. coli is described in Figure 15. The primary structure of the resulting protein has the sequence indicated in the sequence ID No 7. The coding sequence (see sequence ID No. 8) that corresponds to the previous protein design was placed under the control of the promoter? pL in an E. coli expression plasmid. Cloning:
The starting material was the vector PCUZ1 which contains the 117 codons of the C-terminus of the LytA coding region of
Streptococcus pneumoniae and the vector pRIT14518, in which the cDNA of the MAGE-1 gene had previously been subcloned from a plasmid received from Dr. Thierry Boon of the Ludwig Institute. The cloning strategy for the expression of the CLYTA-Mage-1-His protein (see the scheme in Figure 16) included the following steps:
2. - Preparation of the coding sequence module of CLYTA- Mage-1-His
a) The first step was a PCR amplification, designed to flank the CLYTA sequences with the Ndel-Afllll restriction sites. PCR amplification was prmed using the plasmid template PCUZIas and as sensitizers, the sense of the oligonucleotide: 5 'tta aac falls off agg agg ata taa cat atg aaa ggg gga att gta cat tea gac, and the opposite direction to the oligonucleotide: 5 'GCC AGA CAT GTC CAA TTC TGG CCT GTC TGC CAG. This leads to the amplification of a CLYTA sequence of 378 nucleotides long. b) The second step was to link the CLYTA sequences to the MAGE-1-His sequences to generate the coding sequence for the fusion protein. This step included the excision of an Ndel-Afllll Clyta fragment and the insertion in the vector pRIT14518 previously opened by the restriction enzymes Ndl and Ncol (compatible with Ncol and Afllll) and gave rise to the formation of the plasmid pRIT14613. c) Transformation of host strain AR58. d) Selection and characterization of the transformant of E. coli (resistant to KAN), which contains the plasmid pRIT14613 (see Figure 16).
1. - Characterization of the recombinant protein CLYTA-MAGE-1 - His .PR1T14613)
The bacterium was developed in LB medium supplemented with 50 μg / ml of kanamycin at 30 ° C. When the crop had reached OD = 0.3 (at 620 nm), thermal induction was achieved by raising the temperature to 38 ° C. After 4 hours of induction, the cells were harvested, resuspended in PBS and lysed (by disintegration) in a single operation. After centrifugation, the pellet supernatant and the total extract were analyzed by SDS-PAGE. Proteins were visualized on the gels stained with Coomassie B1, where the fusion protein represented approximately 1% of the total proteins of E. coli. The recombinant protein appeared as a single band, with an apparent molecular weight of about 49 kD. The fusion protein was identified by Western blot analysis using polyclonal anti-Mage-1 antibodies.
Reconstitution of the expression unit composed by the long promoter? pL (useful for the induction by nalidixic acid) and the coding sequence of CLYTA-Maqe-1 pRIT14614)
An EcoRI-NCOi restriction fragment containing the long promoter PL and a portion of the CLYTA sequences was prepared from the pRIT DVA6 plasmid and inserted between the EcoRI-NCO sites of the plasmid pRIT14613. The recombinant plasmid pRIT14614 was obtained. The recombinant plasmid pRIT14614 (see Figure 17) encoding the CLYTA-Mage-1-His fusion protein was used to transform E. coli AR120. A candidate strain, resistant to Kan, was selected and characterized.
Characterization of the recombinant protein
Bacteria were developed in LB medium supplemented with 50 mg / ml kanamycin at 30 ° C. When the culture had an OD = 400 (at 620 nm), nalidixic acid was added to a final concentration of 60 mg / ml. After four hours of induction the cells were harvested, they were resuspended in PBS and subjected to lysis by disintegration (CLS-type disintegration "from a single operation"). After centrifugation, the supernatant pellet and the total extract were analyzed by means of SDS-PAGE. The proteins were visualized on gels stained with Coomassie Bleu, where the fusion protein represented approximately 1% of the total E. coli proteins. The fusion protein was identified by Western blot analysis using rabbit anti-Mage-1 polyclonal antibodies. The recombinant protein appeared as a single band, with an apparent molecular weight of approximately 49 kD.
EXAMPLE X
CLYTA - MAGE-3-His
A.- Recombinant tumor rejection antigen: a fusion protein CLYTA-Mage-3-His, where the fusion partner C-lyt A leads to the expression of a soluble protein, acts as an affinity tag and provides an auxiliary T Useful. Preparation of the E. coli strain expressing a CLYTA-Mage-3-His tail of fusion protein. Construction of the expression plasmid pRIT14646 and transformation of the host strain AR 120. Protein design: The design of the Clyta-Mage-3-His fusion protein to be expressed in E. coli is described in figure 18. The primary structure of the resulting protein has the sequence described in sequence ID No. 9, and the coding sequence in sequence ID No. 10. The coding sequence corresponding to the previous protein design was placed under the control of the promoter? pL in an E. coli expression plasmid.
CLONING
The starting material was the vector PCUZ1 containing the 117 codons of the C terminal of the LytA coding region of Streptococcus pneumoniae, described in Gene 43, (1986), page 265-272 and the vector pRIT14426, in which it had previously been subcloned the cDNA of the MAGE-3 gene from a plasmid received from Dr. Tierry Boon, from the Ludwig Institute. The cloning strategy for the expression of the CLYTA-MAGE-3-His protein (see the scheme in Figure 19) included the following steps:
1. - Preparation of the CLYTA-MAGE-3-His coding sequence module
1. 1- The first step was a PCR amplification, designed to flank the CLYTA sequences with the Aflll and Afllll restriction sites. PCR amplification was performed using the plasmid template PCUZIas and as sensitizers the sense of the oligonucleotide: 5 'tta aac falls off ag ag ag a ata ata cat a ggg gga aga gta cat tea gac, and the opposite direction to the oligonucleotide: 'ecc here tgt cea gac tgc tgg cea att ctg gcc tgt ctg cea gtg. This leads to the amplification of a CLYTA sequence of 427 nucleotides long. The above amplified fragment was cloned into a TA cloning vector of Invitrogen, to obtain the intermediate vector pRIT14661. 1.2.- The second step was to link the CLYTA sequences with the MAGE-3-His sequences, to generate the coding sequence for the fusion protein. This step included the removal of a fragment of Clyta Afl ll-Afl-III and the insertion in the vector pRIT14426 previously opened by the restriction enzymes Afl II and Ncol (compatible with Ncol and Aflll) and gave rise to the plasmid pRIT14662.
2. - Reconstitution of the expression unit composed by the long promoter? pL (useful for induction with nalidixic acid) and the coding sequence of CLYTA-Mage-3
A restriction fragment Bgl I-Xba I containing the short promoter pL and the coding sequences of CLYTA-Mage-3-His, was prepared from the plasmid pRIT14662, and inserted between the BglII-Xbal sites of the plasmid TCM67 ( a derivative of pBR322 containing resistance to ampicillin, and the long promoter? pL, described in the international application PCT / EP92 / 01827). Plasmid pRIT14607 was obtained. The recombinant plasmid pRIT14607 encoding the Clyta-Mage-3 His fusion protein was used to transform E. coli AR 120 (Mott et al, 1985, Proc Nati Acad Sci., 82, 88). A candidate strain, resistant to ampicillin, was selected and characterized.
3, - Preparation of plasmid pRIT 14646
Finally, a plasmid similar to pRIT 14607 was constructed, but it had a selection for Kanamycin (pRIT 14646).
CHARACTERIZATION OF THE RECOMBINANT PROTEIN
Bacteria were developed in LB medium supplemented with 50 mg / ml kanamycin at 30 ° C. When the culture had reached OD = 400 (at 600 nm), nalidixic acid was added to a final concentration of 60 mg / ml. After 4 hours of induction, the cells were harvested, resuspended in PBS and lysed by disintegration (CLS disintegration of the "single-operation" type). After centrifugation the pellet supernatant and the total extract were analyzed by SDS-PAGE. The proteins were visualized on gels stained with Coomassie Bleu, where the fusion protein represented approximately 1% of the total of E. coli proteins. The fusion protein was identified by Western blot analysis, using polyclonal rabbit anti-Mage-3 antibodies.
The recombinant protein appeared as a single band, with an apparent molecular weight of about 58 kD.
EXAMPLE XI
PURIFICATION OF RECOMBINANT PROTEIN CLYTA-Maae-3- His
Recombinant bacteria AR120 (pRIT 14646) was developed in a 20 liter fermenter under intermittent feeding conditions at 30 ° C. Expression of the recombinant protein was induced by adding nalidixic acid to a final concentration of 60 mg / ml. The cells were harvested at the end of the fermentation and subjected to lysis at 60 OD / 600, by two passes through a French Press breaker (1.406 kg / cm2). Pellets were formed with the cells lysed for 20 minutes at 15,000 g, at 4 ° C. The supernatant containing the recombinant protein was loaded in DEAE Sepharose CL6B exchange resin (Pharmacia) previously equilibrated in 0.3M NaCl, 20 mM Tris HCl, pH 7.6, which is the regulator A. After a column wash with Regulator A, the fusion protein was eluted by means of 2% choline in regulator A. The antigen-positive fractions were pooled, as revealed by Western blot analysis, using an anti-Mage-3 antibody. The antigen eluted with DEAE was brought to 0.5% of Empigen BB (a hybrid ion detergent) and 0.5M of NaCl, before being loaded onto a column of affinity chromatography with ionic metal, previously equilibrated in 0.5% of Empigen BB, 0.5 M NaCl, 50 mM phosphate buffer pH 7.6 (regulator B). The IMAC column was washed with regulator B until the absorbance at 280 nm reached the baseline. A second wash was carried out in regulator B without Empigen BB (regulator C) in order to remove the detergent, before elution of the antigen by an imidazole gradient from 0 to 250 mM imidazole in regulator C. It was brought together fractions of 0.090-0.250M imidazole were concentrated in a 10 kDa Filtron omega membrane, before dialysis against PBS regulator.
CONCLUSION
It has been shown that the molten protein LPD-IMAGE-3-His is immunogenic in mice, and that this immunogenicity (the proliferation response and the antibody response) can be further increased by the use of the adjuvant described above. The purification can be increased by forming derivatives of the thiols that form the disulfide bonds. It has also been shown that a better response to the antibody was triggered by vaccination with LPD-MAGE-3-His in the presence of the adjuvant. The predominant isotype found in the serum of C57BL / 6, which is IgG2b, suggests that a TH1-type immune response was present. In humans, a clinical patient treated with LPD-MAGE-3-His in an adjuvant-free formulation was melanoma-free.
LIST OF SEQUENCES
(1) GENERAL INFORMATION (i) APPLICANT: SmithKIine Beecham Biologicals (¡i) TITLE OF THE INVENTION: Vaccine (iii) NUMBER OF SEQUENCES: 10 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: SmithKine Beecham (B) ADDRESS: 2 New Horizons Court, Great West Road, B (C) CITY: Middx (D) STATUS: (E) COUNTRY: United Kingdom (F) POSTAL CODE: TW89EP (v) FORM YOU CAN READ ON COMPUTER: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: Compatible with IBM (C) OPERATING SYSTEM: DOS (D) PROGRAM OF APPLICATION: FastSEQ for Windows, version 2.0 (vi) DATA OF THE CURRENT APPLICATION: (A) NUMBER OF APPLICATION: (B) ) DATE OF SUBMISSION: (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (viii) APPORTER / AGENT INFORMATION: (A) NAME: Dalton, Marcus J . (B) REGISTRATION NUMBER: (C) REFERENCE / CASE No. B45126 (ix) TELECOMMUNICATIONS INFORMATION: (A) PHONE: 0181 9745348 (B) TELEFAX: 0181 9756 177 (C) TELEX: (2) INFORMATION FOR SEQ ID No. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 452 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one (D) ) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 1
Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Wing Gly Val Leu
1 5 - 10 15
Wing Gly Cys Ser Ser His Being Being Asn Met Wing Asn Thr Gln Met Ly =
25 30 Ser Asp Lys lie He lie Wing His Arg Gly Wing Ser Gly Tyr Leu Pg§ 35 40 45 Glu His Thr Leu Glu Ser Lys Wing Leu Wing Phe Wing Gln Gln Wing Asp 50 55 60 Tyr Leu Glu Gln Asp Leu Wing Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
He His Asp Hís Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val lie Asp Phe Thr
100 105 110 Leu Lys Glu He Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 115 120 125 Asp Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Glu Leu Glu 130 135 140 Wing Arg Gly Glu Wing Leu Gly Leu Val Gly Ala Gln Ala Pro Ala Thr
145 150 155 160
Glu Glu Gln Glu Wing Wing Being Ser Being Thr Leu Val Glu Val Thr 165 170 175
Leu Gly Glu Val Pro Wing Wing Glu Pro Pro Asp Pro Pro Gln Ser Pro 180 185 190 Gln Gly Wing Ser Ser Leu Pro Thr Met Asn Tyr Pro Leu Trp Ser 195 200 205 Gln Ser Tyr Glu Asp Ser As Asn Gln Glu Glu Glu Gly Pro Ser Thr 210 215 220 Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys Val
225 230 235 240
Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro 245 250 255
Val Thr Lys Wing Glu Met Leu Gly Val Val Gly Asn Trp Gln Tyr 260 265 270 Phe Phe Pro Val He Phe Ser Lys Wing Being Ser Leu Gln Leu Val 275 280 285 Phe Gly He Glu Leu Met Glu Val Asp Pro He Gly His Leu Tyr He 290 295 300 Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn
305 310 315 320
Gln Lie Met Pro Lys Ala Gly Leu Leu He He Val Leu Ala He He 325 330 335
Wing Arg Glu Gly Asp Cys Wing Pro Glu Glu Lys He Trp Glu Glu Leu
340 345 350 Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser He Leu Gly Asp
355 360 365 Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu Glu 370 375 380 Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu Trp
385 390 395 400
Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His His 405 410 415
Met Val Lys He Ser Gly Gly Pro His He Ser Tyr Pro Pro Leu His 420 425 430 Glu Trp Val Leu Arg Glu Glu Glu Glu Thr Ser Gly Gly His His His 435 440 445 His His His 450 (2) INFORMATION FOR SEQ ID No 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1353 base pairs (B) TYPE: nucleic acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear, (ii) TYPE OF MOLECULE : CDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 2:
ATGGATCCñA AAACTTTAGC CCTTTCTTTA TTAGCAGCTG GCGTACTAGC AGGTTGTAGC 60 AGCCATTCAT CAAATATGGC GAATACCCAA ATGAAATCAG ACAAAATCAT TATTGCTCAC 120 CGTGGTGCTA GCGGTTATTT ACCAGAGCAT ACGTTAGAAT CTAAAGCACT TGCGTTTGCA 180 CAACAGGCTG ATTATTTAGA GCAAGATTTA GCAATGACTA AGGATGGTCG TTTAGTGGTT 240 ATTCACGATC ACTTTTTAGA TGGCTTGACT GATGTTGCGA AAAAATTCCC ACATCGTCAT 300 CGTAAAGATG GCCGTTACTA TGTCATCGAC TTTACCTTAA AAGAAATTCA AAGTTTAGAA 360 ATGACAGAAA ACTTTGAAAC CATGGATCTG GAACAGCGTA GTCAGCACTG CAAGCCTGAA. 420 GAAGGCCTTG AGGCCCGAGG AGAGGCCCTG GGCCTGGTGG GTGCGCAGGC TCCTGCTACT 480 GAGGAGCAGG AGGCTGCCTC CTCCTCTTCT ACTCTAGTTG AAGTCACCCT GGGGGAGGTG 540 CCTGCTGCCG AGTCACCAGA TCCTCCCCAG AGTCCTCAGG GAGCCTCCAG CCTCCCCACT 600 ACCATGAACT ACCCTCTCTG GAGCCAATCC TATGAGGACT CCAGCAACCA AGAAGAGGAG 660 GGGCCAAGCA CCTTCCCTGA CCTGGAGTCC GAGTTCCAAG CAGCACTCAG TAGGAAGGTG 720 GCCGAATTGG TTCATTTTCT GCTCCTCAAG TATCGAGCCA GGGAGCCGGT CACAAAGGCA 780 GAAATGCTGG GGAGTGTCGT CGGAAATTGG CAGTATTTCT TTCCTGTGAT CTTCAGCAAA 840 GCTTCCAGTT CCTTGCAGCT GGTCTTTGGC ATCGAGCTGA TGGAAGTGGA CCCCATCGGC 900 CACTTGTACA TCTTTGCCAC CTGCCTGGGC CTCTCCTACG ATGGCCTGCT GGGTGACAAT 960 CAGATCATGC CCAAGGCAGG CCTCCTGATA ATCGTCCTGG CCATAATCGC AAGAGAGGGC 1020 GACTGTGCCC CTGAGGAGAA AATCTGGGAG GAGCTGAGTG TGTTAGAGGT GTTTGAGGGG 1080 AGGGAAGACA GTATCTTGGG GGATCCCAAG AAGCTGCTCA CCCAACATTT CGTGCAGGAA 1140 AACTACCTGG AGTACCGGCA GGTCCCCGGC AGTGATCCTG CATGTTATGA ATTCCTGTGG 1200 GGTCCAAGGG CCCTCGTTGA AACCAGCTAT GTGAAAGTCC TGCACCATAT GGTAAAGATC 1260 AGTGGAGGAC CTCACATTTC CTACCCACCC CTGCATGAGT GGGTTTTGAG AGAGGGGGAA 1320 GAGGGCGGTC ATCACCATCA CCATCACCAT TAA 1353
(2) INFORMATION FOR SEQ ID No.3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1341 base pairs (B) TYPE: nucleic acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear, (ii) TYPE OF MOLECULE: cDNA. (Xi) SEQUENCE DESCRIPTION: SEQ ID No. 3: ATGGATCCAA AAACTTTAGC CCTTTCTTTA TTAGCAGCTG GCGTACTAGC AGGTTGTAGC 60 AGCCATTCAT CAAATATGGC GAATACCCAA ATGAAATCAG ACAAAATCAT TATTGCTCAC 120 CGTGGTGCTA GCGGTTATTT ACCAGAGCAT ACGTTAGAAT CTAAAGCACT TGCGTTTGCA 180 CAACAGGCTG ATTATTTAGA GCAAGATTTA GCAATGACTA AGGATGGTCG TTTAGTGGTT "240
ATTCACGATC ACTTTTTAGA TGGCTTGACT GATGTTGCGA AAAAATTCCC ACATCGTCAT 300 CGTAAAGATG GCCGTTACTA TGTCATCGAC TTTACCTTAA AAGAAATTCA AAGTTTAGAA 360 ATGACAGAAA ACTTTGAAAC CATGGGCTCT CTGGAACAGC GTAGTCTGCA CTGCAAGCCT 420 GAGGAAGCCC TTGAGGCCCA ACAAGAGGCC CTGGGCCTGG TGTGTGTGCA GGCTGCCACC 480 TCCTCCTCCT CTCCTCTGGT CCTGGGCACC CTGGAGGAGG TGCCCACTGC TGGGTCAACA 540 GATCCTCCCC AGAGTCCTCA GGGAGCCTCC GCCTTTCCCA CTACCATCAA CTTCACTCGA 600 CAGAGGCAAC CCAGTGAGGG TTCCAGCAGC CGTGAAGAGG AGGGGCCAAG CACCTCTTGT 660 ATCCTGGAGT CCTTGTTCCG AGCAGTAATC ACTAAGAAGG TGGCTGATTT GGTTGGTTTT 720 CTGCTCCTCA AATATCGAGC CAGGGAGCCA GTCACAAAGG CAGAAATGCT GGAGAGTGTC 780 ATCAAAAATT ACAAGCACTG TTTTCCTGAG ATCTTCGGCA AAGCCTCTGA GTCCTTGCAG 840
CTGGTCTTTG GCATTGACGT GAAGGAAGCA GACCCCACCG GCCACTCCTA TGTCCTTGTC 900 ACCTGCCTAG GTCTCTCCTA TGATGGCCTG CTGGGTGATA ATCAGATCAT GCCCAAGACA 960 GGCTTCCTGA TAATTGTCCT GGTCATGATT GCAATGGAGG GCGGCCATGC TCCTGAGGAG 1020 GAAATCTGGG AGGAGCTGAG TGTGATGGAG GTGTATGATG GGAGGGAGCA CAGTGCCTAT 1080 GGGGAGCCCA GGAAGCTGCT CACCCAÁGAT TTGGTGCAGG AAAAGTACCT GGAGTACCGG 1140 CAGGTGCCGG ACAGTGATCC CGCACGCTAT GAGTTCCTGT GGGGTCCAAG GGCCCTCGCT 1200 GAAACCAGCT ATGTGAAAGT CCTTGAGTAT GTGATCAAGG TCAGTGCAAG AGTTCGCTTT 1260 TTCTTCCCAT CCCTGCGTGA AGCAGCTTTG AGAGAGGAGG AAGAGGGAGT CGGCGGTCAT 1320 CACCATCACC ATCACCATTA A - 1341
(2) INFORMATION FOR SEQ ID No.4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 466 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 4:
Met Asp Pro Lys Thr Leu Ala Leu Ser Leu Leu Ala Wing Gly Val Leu
1 5 10 15
Wing Gly Cys Ser Being His Being Being Asn Met Wing Asn Thr Gln Met Lys 20 25 30 Being Asp Lys He He He Wing His Wing Arg Gly Wing Being Gly Tyr Leu Pro
40 45 Glu His Thr Leu Glu Ser Lys Wing Leu Wing Phe Wing Gln Gln Wing Asp 50 55 60 Tyr Leu Glu Gln Asp Leu Wing Met Thr Lys Asp Gly Arg Leu Val Val
65 70 75 80
He His Asp His Phe Leu Asp Gly Leu Thr Asp Val Ala Lys Lys Phe 85 90 95
Pro His Arg His Arg Lys Asp Gly Arg Tyr Tyr Val He Asp Phe Thr
100 105 110 Leu Lys Glu He Gln Ser Leu Glu Met Thr Glu Asn Phe Glu Thr Met 115 120 125 Gly Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu 130 135 140 Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Wing Thr
145 150 155 160
Be Being Ser Pro Leu Val Leu Gly Thr Leu Glu Val Glu Pro Thr 165 170 175
Wing Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Wing Ser Wing Phe 180 185 190 Pro Thr Thr He Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser 195 200 205 Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys He Leu Glu Ser 210 215 220 Leu Phe Arg Ala Val lie Thr Lys Lys Val Wing Asp Leu Val Gly Phe
225 230 235 240
Leu Leu Leu Lys Tyr Arg Wing Arg Glu Pro Val Thr Lys Ala Glu Met 245 250 255
Leu Glu Ser Val He Lys Asn Tyr Lys His Cys Phe Pro Glu He Phe 260 265 270 Gly Lys Wing Ser Glu Ser Leu Gln Leu Val Phe Gly He Asp Val Lys
- 275 280 285 Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly 290 295 300 Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln He Met Pro Lys Thr
305 310 315 320
Gly Phe Leu He He Val Leu Val Met He Wing Met Glu Gly Gly His 325 330 335
Wing Pro Glu Glu Glu He Trp Glu Glu Leu Ser Val Met Glu Val Tyr 340 345 350 Asp Gly Arg Glu His Ser Wing Tyr Gly Glu Pro Arg Lys Leu Leu Thr
355 360 365 Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp
370 375 380 Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala
385 390 395 400
Glu Thr Ser Tyr Val Lys "Val Leu Glu Tyr Val He Lys Val Ser Wing 405 410 415
Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Wing Leu Arg Glu 420 425 430 Glu Glu Glu Gly Val Gly Gly His His His His His His His His 435 440 445
(2) INFORMATION FOR SEQ ID No. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 404 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 5:
Met Asp Pro Asn Thr Val Being Ser Phe Gln Val Asp Cys Phe Leu Trp
1 5 10 15
His Val Arg Lys Arg Val Wing Asp Gln Glu Leu Gly Asp Wing Pro Phe
25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser
40 45 Thr Leu Gly Leu Asp He Glu Thr Wing Thr Arg Wing Gly Lys Gln He 50 55 60 Val Glu Arg He Leu Lys Glu .Glu Ser Asp Glu Ala Leu Lys Met Thr
65 70 75 80
Met Asp Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu 85 90 95
Glu Wing Arg Gly Glu Wing Leu Gly Leu Val Gly Wing Gln Wing Pro Wing
100 105 110 Thr Glu Glu Gln Glu Wing Wing Being Ser Being Thr Leu Val Glu Val 115 120 125 Thr Leu Gly Glu Val Pro Wing Wing Glu Pro Pro Asp Pro Pro Gln Ser 130 135 140 Pro Gln Gly Wing Being Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp
145 150 155 160
Being Gln Being Tyr Glu Asp Being Being Asn Gln Glu Glu Glu Gly Pro Being 165 170 175
Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys 180 185 190 Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu
195 200 205 Pro Val Thr Lys Wing Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln 210 215 220 Tyr Phe Phe Pro Val He Phe Ser Lys Wing Being Ser Leu Gln Leu
225 230 235 240 Val Phe Gly He Glu Leu Met Glu Val Asp Pro He Gly His Leu Tyr 245 250 255 He Phe Wing Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Glu Asp 260 265 270 Asn Gln He Met Pro Lys Wing Gly Leu Leu He He Le Val Leu Wing He 275 280 285 He Wing Arg Glu Gly Asp Cys Wing Pro Glu Glu Lys He Trp Glu Glu 290 295 300 Leu Ser val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser He Leu Gly 305 310 315 320 Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu 325 330 335 Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu 340 345 350 Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His 355 360 365 His Met Val Lys He Ser Gly Gly Pro His He Ser Tyr Pro Pro Leu 370 375 380 His Glu Trp Val Leu Arg Glu Gly Glu Glu Gly Gly His His Hi = His
(2) INFORMATION FOR SEQ ID No. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1212 base pairs (B) TYPE: nucleic acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: cDNA. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 6:
ATGGATCCAA ACACTGTGTC AAGCTTTCAG GTAGATTGCT TTCTTTGGCA TGTCCGCAAA 60 CGAGTTGCAG ACCAAGAACT AGGTGATGCC CCATTCCTTG ATCGGCTTCG CCGAGATCAG 120 AAATCCCTAA GAGGAAGGGG CAGCACTCTT GGTCTGGACA TCGAGACAGC CACACGTGCT 180 GGAAAGCAGA TAGTGGAGCG GATTCTGAAA GAAGAATCCG ATGAGGCACT TAAAATGACC 240 ATGGATCTGG AACAGCGTAG TCAGCACTGC AAGCCTGAAG AAGGCCTTGA GGCCCGAGGA 300 GAGGCCCTGG GCCTGGTGGG TGCGCAGGCT CCTGCTACTG AGGAGCAGGA GGCTGCCTCC 360 TCCTCTTCTA CTCTAGTTGA AGTCACCCTG GGGGAGGTGC CTGCTGCCGA GTCACCAGAT 420 CCTCCCCAGA GTCCTCAGGG AGCCTCCAGC CTCCCCACTA CCATGAACTA CCCTCTCTGG 480 AGCCñATCCT ATGAGGACTC CAGCAACCAA GAAGAGGAGG GGCCAAGCAC CTTCCCTGAC 540 CTGGAGTCCG AGTTCCAAGC AGCACTCAGT AGGAAGGTGG CCGAATTGGT TCATTTTCTG 600 CTCCTCAAGT ATCGAGCCAG GGAGCCGGTC ACAAAGGCAG AAATGCTGGG GAGTGTCGTC 660 GGAAATTGGC AGTATTTCTT TCCTGTGATC TTCAGCAAAG CTTCCAGTTC CTTGCAGCTG 720 GTCTTTGGCA TCGAGCTGAT GGAAGTGGAC CCCATCGGCC ACTTGTACAT CTTTGCCACC 780 TGCCTGGGCC TCTCCTACGA TGGCCTGCTG GGTGACAATC AGATCATGCC CAAGGCAGGC 840 CTCCTGATAA TCGTCCTGG C CATAATCGCA AGAGAGGGCG ACTGTGCCCC TGAGGAGAAA 900 ATCTGGGAGG AGCTGAGTG GTTAGAGGTG TTTGAGGGGA GGGAAGACAG TATCTTGGGG 960 GATCCCAAGA AGCTGCTCAC CCAACATTTC GTGCAGGAAA ACTACCTGGA GTACCGGCAG 1020 GTCCCCGGCA GTGATCCTGC ATGTTATGAA TTCCTGTGGG GTCCAAGGGC CCTCGTTGAA 1080 ACCAGCTATG TGAAAGTCCT GCACCATATG GTAAAGATCA GTGGAGGACC TCACATTTCC 1140 TACCCACCCC TGCATGAGTG GGTTTTGAGA GAGGGGGAAG AGGGCGGTCA TCACCATCAC 1200 CATCACCATT AA 1212 (2) INFORMATION FOR SEQ ID No. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 445 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: protein. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 7:
Met Lys Gly Gly He Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys He Asn Gly Thr Trp Tvr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30 Met Leu Wing Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp 35 40 45 Phe Asp Asn Ser Gly Glu Met Wing Thr Gly Trp Lys Lys He Wing Asp 50 55 60 Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Wing Met Lys Thr Gly Trp Val 65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Wing Lys Glu Gly Wing Met 85 90 95
Val Ser Asn Wing Phe He Gln Ser Wing Asp Gly Thr Gly Trp Tyr Tyr 100 105 110
Leu Lys Pro Asp Gly Thr Leu Wing Asp Arg Pro Glu Leu Asp Met Gly
115 120 125 Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Wing Leu Glu 130 135 140 Wing Gln Gln Glu Wing Leu Gly Leu Val Cys Val Gln Wing Wing Thr Ser
145 150 155 160
Being Ser Pro Leu Val Leu Gly Thr Leu Glu Val Glu Pro Thr Ala 165 170 175
Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Wing Ser Wing Phe Pro 180 185 190 Thr Thr He Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser Ser 195 200 205 Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys He Leu Glu Ser Leu 210 215 220 Phe Arg Ala Val He Thr Lys Lys Val Wing Asp Leu Val Gly Phe Leu
225 230 235 240 Leu Leu Lys Tyr Arg Wing Arg Glu Pro Val Thr Lys Wing Glu Met Leu 245 250 255 Glu Ser Val He Lys Asn Tyr Lys His Cys Phe Pro Glu He Phe Gly 260 265 270 Lys Wing Ser Glu Ser Leu Gln Leu Val Phe Gly He Asp Val Lys Glu 275 280 285 Wing Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly Leu 290 295 300 Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln He Met Pro Lys Thr Gly
305 310 315 320
Phe Leu He He Val Leu Val Met He Wing Met Glu Gly Gly His Wing 325 330 335 Pro Glu Glu Glu He Trp Glu Glu Leu Ser Val Met Glu Val Tyr A = p 340 345 350 Gly Arg Glu His Ser Wing Tyr Gly Glu Pro Arg Lys Leu Leu Thr Gln 355 360 365 Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp Ser 370 375 380 Asp Pro Wing Arg Tyr Glu Phe Leu Trp Gly Pro Arg Wing Leu Wing Glu
385 390 395 400
Thr Ser Tyr Val Lys Val Leu Glu Tyr Val He Lys Val Ser Wing Arg 405 410 415 Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Wing Leu Arg Glu Glu 420 425 430 Glu Glu Gly Val Gly Gly His His His His His His His His 435 440 445
(2) INFORMATION FOR SEQ ID No. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1338 base pairs (B) TYPE: nucleic acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear, (¡i) TYPE OF MOLECULE: cDNA. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 8:
ATGAAAGGGG GAATTGTACA TTCAGACGGC TCTTATCCAA AAGACAAGTT TGAGAAAATC 60 AATGGCACTT GGTACTACTT TGACAGTTCA GGCTATATGC TTGCAGACCG CTGGAGGAAG 120 CACACAGACG GCAACTGGTA CTGGTTCGAC AACTCAGGCG AAATGGCTAC AGGCTGGAAG 180 AAAATCGCTG ATAAGTGGTA CTATTTCAAC GAAGAAGGTG CCATGAAGAC AGGCTGGGTC 240 AAGTACAAGG ACACTTGGTA CTACTTAGAC GCTAAAGAAG GCGCCATGGT ATCAAATGCC 300 TTTATCCAGT CAGCGGACGG AACAGGCTGG TACTACCTCA AACCAGACGG AACACTGGCA 360 GACAGGCCAG AATTGGACAT GGGCTCTCTG GAACAGCGTA GTCTGCACTG CAAGCCTGAG 420 GAAGCCCTTG AGGCCCAACA AGAGGCCCTG GGCCTGGTGT GTGTGCAGGC TGCCACCTCC 480 TCCTCCTCTC CTCTGGTCCT GGGCACCCTG GAGGAGGTGC CCACTGCTGG GTCAACAGAT 540 CCTCCCCAGA GTCCTCAGGG AGCCTCCGCC TTTCCCACTA CCATCAACTT CACTCGACAG 600 AGGCAACCCA GTGAGGGTTC CAGCAGCCGT GAAGAGGAGG GGCCAAGCAC CTCTTGTATC 660 CTGGAGTCCT TGTTCCGAGC AGTAATCACT AAGAAGGTGG CTGATTTGGT TGGTTTTCTG 720 CTCCTCAAAT ATCGAGCCAG GGAGCCAGTC ACAAAGGCAG AAATGCTGGA GAGTGTCATC 780 AAAAATTACA AGCACTGTTT TCCTGAGATC TTCGGCAAAG CCTCTGAGTC CTTGCAGCTG 840 GTCTTTGGCA TTGACGTGA A GGAAGCAGAC CCCACCGGCC ACTCCTATGT CCTTGTCACC 900 TGCCTAGGTC TCTCCTATGA TGGCCTGCTG GGTGATAATC AGATCATGCC CAAGACAGGC 960 TTCCTGATAA TTGTCCTGGT CATGATTGCA ATGGAGGGCG GCCATGCTCC TGAGGAGGAA 1020 ATCTGGGAGG AGCTGAGTGT GATGGAGGTG TATGATGGGA GGGAGCACAG TGCCTATGGG 1080 GAGCCCAGGA AGCTGCTCAC CCAAGATTTG GTGCAGGAAA AGTACCTGGA GTACCGGCAG 1140 GTGCCGGACA GTGATCCCGC ACGCTATGAG TTCCTGTGGG GTCCAAGGGC CCTCGCTGAA 1200 ACCAGCTATG TGAAAGTCCT TGAGTATGTG ATCAAGGTCA GTGCAAGAGT TCGCTTTTTC 1260 TTCCCATCCC TGCGTGAAGC AGCTTTGAGA GAGGAGGAAG AGGGAGTCGG CGGTCATCAC 1320 CATCACCATC ACCATTAA 1338
(2) INFORMATION FOR SEQ ID No.9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 454 amino acids (B) TYPE: amino acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear. (I) TYPE OF MOLECULE: protein. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 9:
Met Lys Gly Gly He Val His Ser Asp Gly Ser Tyr Pro Lys Asp Lys
1 5 10 15
Phe Glu Lys He Asn Gly Thr Trp Tyr Tyr Phe Asp Ser Ser Gly Tyr 20 25 30 Met Leu Wing Asp Arg Trp Arg Lys His Thr Asp Gly Asn Trp Tyr Trp
40 45 Phe Asp Asn Ser Gly Glu Met Wing _hr Gly Trp Lys Lys He Wing Asp
50 55 60 Lys Trp Tyr Tyr Phe Asn Glu Glu Gly Wing Met Lys Thr Gly Trp Val
65 70 75 80
Lys Tyr Lys Asp Thr Trp Tyr Tyr Leu Asp Wing Lys Glu Gly Wing Met 85 90 95
Val Ser Asn Wing Phe He Gln Ser Wing Asp Gly Thr Gly Trp Tyr Tyr 100 105 110 Leu Lys Pro Asp Gly Thr Leu Wing Asp Arg Pro Glu Leu Wing Being Met
115 120 125 Leu Asp Met Asp Leu Glu Gln Arg Ser Gln His Cvs Lys Pro Glu Glu
130 135 140 Gly Leu Glu Wing Arg Gly Glu Wing Leu Gly Leu al Gly Wing Gln Ala
145 150 155 160
Pro Wing Thr Glu Glu Gln Glu Wing Wing Being Being Ser Thr Leu Val 165 170 175
Glu Val Thr Leu Gly Glu Val Pro Ala Wing Glu Pro Pro Asp Pro Pro 180 185 190 Gln Ser Pro Gln Gly Wing Being Ser Leu Pro Thr Thr Met Asn Tyr Pro 195 200 205 Leu Trp Ser Gln Ser Tyr Glu Asp Being Ser Asn Gln Glu Glu Glu Gly
210 215 220 Pro Ser Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser
225 230 235 240
Arg Lys Val Wing Glu Leu Val HiS Phe Leu Leu Leu Lys Tyr Arg Wing 245 250 255
Arg Glu Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn 260 265 270 Trp Gln Tyr Phe Phe Pro Val He Phe Ser Lys Wing Ser Ser Leu 275 280 285 Gln Leu Val Phe Gly He Glu Leu Met Glu Val Asp Pro He Gly His
(2) INFORMATION FOR SEQ ID No. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1362 base pairs (B) TYPE: nucleic acid (C) No. OF FILAMENTS: one only (D) TOPOLOGY: linear. (ii) TYPE OF MOLECULE: cDNA.
(xi) D ESCRI P C IO N D E LA S E C O N C E: S EQ I D N o. 1 0:
ATGAAAGGGG GAATTGTACA TTCAGACGGC TCTTATCCAA AAGACAAGTT TGAGAAAATC 60 AATGGCACTT GGTACTACTT TGACAGTTCA GGCTATATGC TTGCAGACCG CTGGAGGAAG 120 CACACAGACG GCAACTGGTA CTGGTTCGAC AACTCAGGCG AAATGGCTAC AGGCTGGAAG 180 AAAATCGCTG ATAAGTGGTA CTATTTCAAC GAAGAAGGTG CCATGAAGAC AGGCTGGGTC 240 AAGTACAAGG ACACTTGGTA CTACTTAGAC GCTAAAGAAG GCGCCATGGT ATCAAATGCC 300 TTTATCCAGT CAGCGGACGG AACAGGCTGG TACTACCTCA AACCAGACGG AACACTGGCA 360 GACAGGCCAG AA.TTGGCCAG CATGCTGGAC ATGGATCTGG AACAGCGTAG TCAGCACTGC 420 AAGCCTGAAG AAGGCCTTGA GGCCCGAGGA GAGGCCCTGG GCCTGGTGGG TGCGCAGGCT 480 CCTGCTACTG AGGAGCAGGA GGCTGCCTCC TCCTCTTCTA CTCTAGTTGA AGTCACCCTG 540 GGGGAGGTGC CTGCTGCCGA GTCACCAGAT CCTCCCCAGA GTCCTCAGGG AGCCTCCAGC 600 CTCCCCACTA CCATGAACTA CCCTCTCTGG AGCCAATCCT ATGAGGACTC CAGCAACCAA 660 GAAGAGGAGG GGCCAAGCAC CTTCCCTGAC CTGGAGTCTG AGTTCCAAGC AGCACTCAGT 720 AGGAAGGTGG CCAAGTTGGT TCATTTTCTG CTCCTCAAGT ATCGAGCCAG GGAGCCGGTC 780 ACAAAGGCAG AAATGCTGGG GAGTGTCGTC GGAAATTGGC AGTACTTCTT TCCTGTGATC 840 TTCAGCAAAG CTTCCGATT C CTTGCAGCTG GTCTTTGGCA TCGAGCTGAT GGAAGTGGAC 900 CCCATCGGCC ACGTGTACAT CTTTGCCACC TGCCTGGGCC TCTCCTACGA TGGCCTGCTG 960 GGTGACAATC AGATCATGCC CAAGACAGGC TTCCTGATAA TCATCCTGGC CATAATCGCA 1020 AAAGAGGGCG ACTGTGCCCC TGAGGAGAAA ATCTGGGAGG AGCTGAGTGT GTTAGAGGTG 1080 TTTGAGGGGA GGGAAGACAG TATCTTCGGG GATCCCAAGA AGCTGCTCAC CCAATATTTC 1140 GTGCAGGAAA ACTACCTGGA GTACCGGCAG GTCCCCGGCA GTGATCCTGC ATGCTATGAG 1200 TTCCTGTGGG GTCCAAGGGC CCTCATTGAA ACCAGCTATG TGAAAGTCCT GCACCATATG 1260 GTAAAGATCA GTGGAGGACC TCGCATTTCC TACCCACTCC TGCATGAGTG GGCTTTGAGA 1320 GAGGGGGAAG AGGGCGGTCA TCACCATCAC CATCACCATT AA 1362
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Claims (17)
1. - An antigenic derivative associated with tumor, of the MAGE family, characterized in that said derivative is selected from the group: • MAGE antigen comprising thiol residues formed to derivatives; • a MAGE fusion protein, expressed recombinantly, in which the fusion partner is selected from protein D or a fragment thereof; the NS1 protein of the influenza or a fragment of it, or C-lyta of Streptococcus pneumoniae or a fragment of it.
2. An antigen according to claim 1, further characterized in that the free thiols formed adrenergized are carboxyamidated or carboxymethylated.
3. An antigen according to claim 1 or 2, further characterized in that the antigen comprises an affinity tag.
4. An antigen according to any of claims 1 to 3, further characterized in that the protein D or its fragment are lipidated.
5. An antigen according to any of claims 1 to 4, further characterized in that the MAGE protein is selected from the group: MAGE A1, MAGE A2, MAGE A3, MAGE A4, MAGE A5, MAGE A6, MAGE A7, MAGE A8 , MAGE A9, MAGE A10, MAGE A11, MAGE A12, MAGE B1, MAGE B2, MAGE B3, MAGE B4, MAGE C1 and MAGE C2.
6. A nucleic acid sequence characterized in that it encodes a fusion protein as claimed in any of claims 1 to 5.
7. A vector, characterized in that it comprises a nucleic acid of claim 6.
8.- A transformed host with a nucleic acid of claim 6 or with a vector of claim 7.
9. A vaccine, characterized in that it contains a protein as claimed in any of claims 1 to 5.
10. A vaccine according to claim 9, further characterized in that it additionally comprises an adjuvant and / or cytokine or chemokine immunostimulants.
11. A vaccine according to claim 9 or 10, further characterized in that the protein is presented in an oil-in-water emulsion vehicle.
12. A vaccine according to claim 10 or 11, further characterized in that the adjuvant comprises 3D-MPL, QS21 or the CpG oligonucleotide.
13. A vaccine as claimed herein, further characterized by additionally comprising one or more additional antigens.
14. A vaccine as claimed herein, further characterized because it is used in medicine.
15. The use of a protein or a nucleic acid as claimed herein, for the manufacture of a vaccine to immunotherapeutically treat a patient suffering from melanomas or other tumors associated with MAGE.
16. A process for purifying a MAGE protein or a derivative thereof, characterized in that it comprises reducing disulfide ligatures, blocking the resulting free thiol group with a blocking group, and subjecting the resulting derivative to one or more purification steps by chromatography.
17. A process for the production of a vaccine, characterized in that it comprises the steps of purifying a MAGE protein or a derivative thereof, by the process of claim 19, and formulating the resulting protein as a vaccine.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9802543.0 | 1998-02-05 | ||
GB9802650.3 | 1998-02-06 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA00007677A true MXPA00007677A (en) | 2001-07-03 |
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