HK1063725A - Vaccine composition containing transforming growth factor alpha - Google Patents

Description

Vaccine composition containing transforming growth factor alpha

Technical Field

The present invention relates to the fields of immunology and human medicine, in particular to a vaccine formulation capable of eliciting an immune castration response against autologous TGF α. The vaccine can be used for treating certain cancers and other TGF alpha related diseases.

Prior Art

Transforming growth factor (TGF. alpha.) is a 50 amino acid polypeptide originally isolated from the conditioned medium of retroviral transformed cells. Initially it was considered to be a molecule capable of competing with Epidermal Growth Factor (EGF) for binding to EGF-R. However, anti-EGF antibodies do not recognize TGF-alpha (Todaro et al (1976), Nature 264, 26-31). Thus, the two growth factors are two immunologically distinct entities.

TGF α belongs to the EGF family. Structurally and functionally related proteins make up this family. Other members of this family are EGF, Amphiregulin (AR), criptol (CR1), heparin-binding EGF, betacellulin, epiregulin. On the other hand, the poxviridae family includes EGF related proteins. The most representative of these is vaccinia Virus Growth Factor (VGF).

All these molecules bind and activate EGF-R, which is why they are considered as ligands for this receptor. This system plays a role in the growth of normal and tumor cells. EGF-R is a 170kD glycoprotein whose gene has been cloned and sequenced. The intracellular domain of the receptor is associated with tyrosine kinase protein activity. These proteins share structural homology with the v-erb-B oncogene product, which has been shown to be involved in tumor transformation (Heldin C.H, (1984), Cell 37, 9-20.)

TGF α is synthesized as a 160 amino acid transmembrane precursor (pro TGF α). Mature TGF α, a soluble form of 50 amino acids, is released by proteolytic cleavage. Human TGF α (hTGF α) shows 43% amino acid sequence identity to human egf (hegf) and 93% identity to mouse or rat TGF α. Furthermore, their biological effects are not species-specific.

A number of laboratory works have demonstrated the ability of TGF α to regulate proliferation, migration and differentiation of cultured cells (Carpenter and Wahl, (1990), Springer-Verlag, Berlin, pp 69-171).

TGF-alpha is the most widely occurring EGF-R ligand. It is expressed in normal tissues during embryogenesis and in normal and tumor tissues of adults. However, no major pathological defect was observed in mice knocked out for TGF α, and these mice could survive and proliferate (Bruce Mann and colleagues (1993), Cell, 73, 249-261).

During tumorigenesis, deregulation of the EGF-R activated paracrine and autocrine processes is caused by upregulation of growth factor expression or hypersynthesis or mutation of its receptor.

High levels of EGF-R were detected in epithelial tumors. In many cases, overexpression of this receptor is predictive of a poor prognosis.

Induction of TGF α is common in tumor transformation. In fact, numerous studies have shown that the molecule is overexpressed in epithelial tumors in various locations, including breast, lung, brain, liver, prostate, bladder, gastrointestinal tract, colon, reproductive (ovarian) and endocrine tissues, among others.

Although the mechanism by which TGF α induces tumorigenesis is not well defined, several reports have been made to link the overexpression of this growth factor to the staging of tumors, patient survival, and other tumor markers. In addition, some researchers have demonstrated their relationship to other oncogenes, such as c-myc in liver cancer. TGF α is also a target for the von Hippel-Lindau tumor suppressor gene (VHL) (summarized in Lee et al, (1996), Growth Factors and Cytokines in health and Disease, Vol.1B, 277-.

Although TGF α and EGF bind to the same receptor with similar affinities, TGF α is generally more potent than EGF, and in some cases its effect is stated to be stronger and/or more durable (Barrandon and Green (1987), Cell, 50, 1131-1137). TGF-alpha and EGF-R are reported to preferentially recycle back to the Cell surface for the internalized TGF-alpha/EGF-R complex, whereas both components are effectively degraded when the EGF/EGF-R complex is internalized into the same Cell type (Ebner and Derynck (1991), Cell Regul.2, 599-612). These results indicate that the differences in biological activity of each growth factor may be due to different intracellular transport mechanisms.

TGF α, on the other hand, is a stronger angiogenic factor than EGF (Schreiber et al, (1986), Science 232, 1250-1253).

With respect to expression in tumors, there is evidence that EGF precursors are present in the cell membrane of certain epithelial tumors, but TGF α is most expressed in epithelial tumors, and in contrast to EGF, it acts through an autocrine loop with EGF-R. On the other hand, our central results show that TGF α is expressed in biopsies of some epithelial tumors without EGF (breast ductal carcinoma, laryngeal carcinoma), but other tumors present more EGF than TGF α (non-small cell lung cancer (NSCLC)). These results indicate that growth factors have different effects in tumor biology of different tumor cells.

All the evidence accumulated over the years about the relationship between the EGF-R/EGF-R ligand system and cancer makes this system an extremely attractive target in cancer immunotherapy.

Our group's previous results demonstrated that an EGF based vaccine could be developed for active cancer immunotherapy. In fact, preclinical and clinical evidence has been obtained regarding immunogenicity and low toxicity resulting from vaccination with hEGF coupled to a carrier protein (Gonz a lez et al (1996), Vaccine Research 5(4), 233-.

Preclinical studies have shown that hEGF-immunized mice in adjuvant can increase the survival of Ehrlich Ascites Tumor (EAT) -transplanted mice (Gonz a lez et al (1996), vaccine research 5(4), 233-.

A fusion protein of hEGF and P64K was prepared. The protein contains the hEGF sequence inserted between the 45/46 amino acids of P64K. The fusion protein is used to immunize mice to elicit a specific humoral immune response against hEGF. The immune response elicited extended the life of mice loaded with EAT (Gonz lez and colleagues (1997), Vaccine Research 6(2), 91-100).

In both clinical trials in patients with NSCLC, an increased trend was observed in the survival of vaccinated patients compared to the historical controls. Patients with high antibody responses against hEGF increased significantly (Gonz-lez et al (1998), Annals of oncology9, 1-5).

Typically, EGF vaccination does not generate specific antibody responses against TGF-alpha. However, evidence has been obtained that only some mice produce low levels of anti-EGF antibodies after vaccination of mouse models with immunogenic formulations containing TGF α. This antibody response can in some cases block EGF binding to its receptor in vitro. However, the level of anti-EGF antibody obtained is not sufficient to produce an effective EGF immunodepletion reaction and to exert an effect in the anti-tumour action.

Since each of these growth factors acts differently between individual tumors and/or primary tumors and their metastases, a vaccine combining the two main ligands TGF α and EGF for EGF-R generally has a better anti-tumor effect against epithelial tumors.

Prior to the present invention, there has not been any treatment for active immunotherapy of cancer using vaccine formulations containing hTGFa or any derivative, or its combination with EGF, the other ligand for EGF-R.

Disclosure of Invention

The present invention provides a vaccine composition comprising TGF α or any derivative of any source thereof, genetically linked (fusion protein) to a carrier protein or chemically coupled, which is capable of inhibiting the growth of epithelial tumors without adverse side effects. This effect is achieved by the growth factor immune castration mechanism. The invention also claims a vaccine composition comprising hTGFa in combination with hEGF or any derivative and a carrier protein.

The vaccine composition may be used to treat TGF alpha or TGF alpha/EGF dependent epithelial tumours, or any other disease associated with TGF alpha, such as psoriasis (Kapp et al (1993) JDermatol Sci, Jun; 5 (3): 133-42).

In the specification of TGF α, any TGF α -derived fragment having the same immunological properties and/or similar effect as the original molecule is included. Those derivatives, including but not excluding others: original substitution of amino acids, alteration of specific amino acids to increase stability and/or activity, chemical modification, and the like.

More particularly, the present invention provides a vaccine composition capable of eliciting an autoimmune castration response from TGF-alpha, which may be used in the treatment of certain cancers and other TGF-alpha related diseases.

In another aspect the invention includes the use of a vaccine formulation consisting of a combination of TGF α and EGF. The vaccine is useful for the treatment of tumors that depend on both growth factors during their pathogenesis.

1-immunogenic formulation:

the hTGFa comprised by the vaccine formulation used in the present invention is either coupled to a carrier protein by genetic engineering methods (fusion protein) or chemically bound thereto. The hTGFa used in any of these immunogenic agents may be recombinant, synthetic or derived from natural sources. Different proteins can be used as carriers. Examples of carrier proteins that can be used are: tetanus toxoid, KLH, P64K protein from neisseria meningitidis and others. The optimal amount of hTGFa in the vaccine formulation fluctuates between 5. mu.g and 1000. mu.g per dose.

On the other hand, a vaccine preparation containing a combination of hTGFa and hEGF was used (National drug Registration Office, Office of National Registration of medicine, HEBERMIN Not 1266).

In the specification of TGF α or EGF, any TGF α or EGF-derived fragment having the same immunological properties and/or similar effects as the original molecule is included. Those derivatives include, but do not exclude others: original substitutions of amino acids, changes to specific amino acids to increase stability and/or activity, chemical modifications, and others.

A) Obtaining fusion protein TGF alpha-carrier protein by a genetic engineering method:

the gene (500bp) encoding hTGFa was amplified by Polymerase Chain Reaction (PCR) using specific primers. The resulting DNA fragment is digested and cloned at a specific site into an expression vector containing a gene encoding a carrier protein. The protein obtained contains one or more copies of both molecules. Mammalian cells, bacteria or yeast can be used as expression vectors. The vector may also include 6 histidines at the N-terminus of the carrier protein. The plasmids obtained were confirmed by restriction analysis on agarose gels, DNA sequencing using Sequenase2.0(Amersham-USB) and finally analysis of any E.coli expression strain expressing fusion protein by Western blotting technique using specific monoclonal antibodies against hTGFa (R & DSystem). To obtain the protein, the bacterial cell wall is disrupted using a strong disruption method, and then the protein is purified using a combination of ammonium sulfate differential precipitation and chromatography. Finally, the protein is filtered under sterile conditions and stored at-20 ℃ or lyophilized and stored at 4 ℃ until later use.

B) Obtaining a chemical conjugate containing hTGF alpha:

different preparations containing hTGF α bound to different carrier proteins (e.g., P64K) were obtained. Any chemical bonding method may be used. The preferred chemistry is north american patent u.s.pat, not.4,302,386; methods using EMCS materials are described in Lee et al, 1981.

Alternatively, glutaraldehyde bonding methods may be used. Briefly, these two or three molecules at a concentration of 1mg/ml in the final solution were mixed with 0.05% glutaraldehyde (in the total solution). The mixture was incubated at room temperature for 1 hour, then 1X/10 mM MgCl in PBS₂And (5) dialyzing the solution. Finally, the mixture was dialyzed with PBS 1X overnight at 4 ℃. The immunogenic preparation is filtered under sterile conditions and stored at 4 ℃ until use.

C) Obtaining the vaccine combining the hTGF alpha and the hEGF.

Obtaining a vaccine that binds the two main ligands of EGF-R can be done in different ways:

1-mixing two vaccines containing hTGGF alpha or hEGF separately associated with a carrier protein at 1: 1 ratio at the time of injection. Fusion proteins or chemical conjugates of each growth factor with a carrier protein may be used for this purpose. The optimal amount of hTGFa and hEGF in the vaccine formulation fluctuates between 5 μ g and 1000 μ g per dose.

2-obtaining a similar genetic construct according to section a, which contains two growth factors hTGF α and hEGF, or any derivative thereof in combination.

3-use of the method described in section B to obtain a chemical conjugate comprising a combination of hTGFa and hEGF or any derivative thereof and a carrier protein by chemical means.

D) Obtaining an immunogenic preparation:

to obtain the desired immunogenic effect of the vaccine composition, it may be convenient to use a suitable adjuvant and to select a route of administration such that the vaccine formulation exhibits high immunogenicity.

The vaccine composition of the present invention is prepared by two specific ways:

1) using Al (OH)₃As an adjuvant to obtain an aqueous solution, the vaccine formulation is adsorbed onto the compound. For this purpose, 2 to 5mg of Al (OH) are bound with TGF alpha equivalent to 5 to 1000. mu.g in different formulations₃The above. The preparation was shaken for 1 hour and the final solution was stored at 4 ℃ until later use.

2) An incomplete freund's adjuvant was used to form an aqueous/oil or oil/water emulsion. The amount of fusion protein or chemical conjugate and adjuvant in the final formulation ranges from 40/60 to 60/40 (vol/vol). The volume used depends on the final emulsion desired to be obtained. Adjuvant was added prior to immunization and the formulation was shaken at room temperature for 10 to 30 minutes.

The final volume of each immunogenic formulation covers the appropriate range for the respective route of administration.

For the case of the combination vaccine prepared immediately upon injection as described in section C, the two vaccines mixed with the appropriate adjuvant are mixed by shaking and injected or injected separately as described previously.

The vaccine composition may be administered by a variety of routes: intramuscular, subcutaneous, intranasal, and intradermal.

Examples

Example 1: the DNA fragment encoding mature TGF α was obtained by Polymerase Chain Reaction (PCR).

The gene encoding hTGFa was amplified by PCR using PSK/TGF alpha vector (CIGB, Gouba) as template. This plasmid contains the hTGFa complementary DNA (cDNA) cloned into the EcoR V site of the commercial vector pBluescript KS- (Stragene). The specific primers were used to amplify sequences encoding mature TGF α (50 amino acids long (fig. 1)):

n-terminal: 5' -GCTCTAGAAGTGGTGTCCCATTTTAATGAC-3’

(underlined, XbaI restriction sites)

C terminal: 5' -CGGAATTCGCCAGGAGGTCCGCATGCTCAC-3’

(underlined, EcoRI restriction sites)

Briefly, 200ng of PSKTGF α was used in 75 μ L of the mixture, which contained: 500ng each of the specific primers, 200mM each of a mixture of deoxynucleotide triphosphates, 25mM MgCl₂And 100 units of Taq-polymerase (Promega) dissolved in a buffer solution supplied by Promega. 30 cycles of denaturation (94 ℃ for 1 min), annealing (60 ℃ for 1 min) and extension (72 ℃ for 30 sec) were performed. Before the first cycle, the DNA was denatured for 4 minutes; after the last cycle, the final extension was carried out for 2 minutes.

The PCR products were electrophoretically separated on a low melting point (LGT) agarose gel and the amplified gene was purified by phenol extraction and digested with XbaI and EcoRI enzymes (NEB, USES) according to conventional procedures. Prepared according to this protocol is a gene fragment encoding mature TGF α.

Example 2: obtaining the expression vector of the fusion protein TGF alpha-P64K.

The expression vector pM92 (CIGB, cuba) was used. This plasmid contains the lpdA gene encoding the P64K protein of Neisseria meningitidis (strain B385), which is under the control of the E.coli tryptophan operon promoter (ptrp) and the phage T4 transcription terminator (tT 4). pM92 encodes ampicillin and kanamycin antibiotic resistance. Dam-E.coli strain (GC-366) was transformed with pM92 and the plasmid was purified using a commercial kit (Quiagen) according to the manufacturer's protocol. The PM92 vector was digested and purified from LGT sepharose. The PM92 vector was then ligated with the previously prepared cDNA of mature hTGGF alpha using T4 ligase (Gibco BRL). The resulting plasmid, pMTGF α, encodes a fusion protein containing hTGGF α inserted between the 45/46 amino acids of P64K. The recombinant plasmid was confirmed by agarose gel restriction analysis, DNA sequencing using Sequenase2.0(Amersham-USB), and finally analysis of the expression of the fusion protein in E.coli strain MM299 by Western blotting technique using a monoclonal antibody specific for hTGFa (R & D System). FIG. 2 shows a schematic of the process of obtaining the expression vector pMTGF α. The vector encodes the fusion protein TGF alpha-P64K.

Example 3: an expression vector (pMHisTGF. alpha.) for the fusion protein TGF. alpha. -P64K having 6 histidines at the N-terminus was obtained.

The expression vector pMHisTGF α was obtained according to the same protocol as described in the previous example, starting with pM224, which included a fragment encoding the N-terminal 6 histidines of P64K. Due to the Cu-carried²⁺Or other metals, the 6 histidine tags show advantages in protein purification.

Example 4: the fusion protein (TGF alpha-P64K) was purified.

Coli (MM299 strain) expressing the fusion protein TGF-alpha-P64K was grown in LBA medium (10g/L tryptone, 5g/L yeast extract, 10g/L NaCl and 50mg/L ampicillin) at 37 ℃ for 10 hours. After collection of the cells, all steps were performed at 0-4 ℃. In a French press at 1500kg/cm²The bacteria were disrupted and centrifuged at 11,000 Xg for 30 minutes at high speed to remove insoluble fractions. The first purification step uses a 40% ammonium sulfate precipitation to remove a portion of the E.coli protein. The resulting precipitate was further centrifuged at 11,000 Xg at 4 ℃ for 30 minutes to remove the precipitate. The supernatant was fractionated by hydrophobic interaction chromatography (TSK-butil, Pharmacia, Sweden) using a gradient of ammonium sulphate decreasing from 40% to 0% in Tris-Cl buffer at pH 7.2 containing 0.15M NaCl. The resulting sample was then separated by gel filtration through a G200 column (Pharmacia) equilibrated at 1 x with PBS to a final purity of greater than 95%. Protein concentrations were determined by colorimetric methods as described by Lowry et al (1951) J.biol.chem.191, 495-498. Using antibodiesP64K and antibodies specific for TGF α, the fusion protein was characterized by western blotting techniques.

Example 5: the fusion protein (HisTGF alpha-P64K) was purified.

Coli (MM299 strain) expressing the fusion protein TGF-alpha-P64K was grown in LBA medium (10g/L tryptone, 5g/L yeast extract, 10g/L NaCl and 50mg/L ampicillin) at 37 ℃ for 10 hours. After collection of the cells, all steps were performed at 0-4 ℃. In a French press at 1500kg/cm²The bacteria were disrupted and centrifuged at 11,000 Xg for 30 minutes at high speed to remove insoluble fractions. The first purification step uses a 40% ammonium sulfate precipitation to remove a portion of the E.coli protein. The resulting precipitate was removed by further centrifugation at 11,000 Xg for 30 minutes at 4 ℃. Due to the presence of 6 histidines in the protein, the supernatant was fractionated by chelate affinity chromatography (chemical Sepharose Fast Flow, Pharmacia, Sweden) using an increasing imidazole gradient from 25mM to 500mM in Tris-Cl buffer at pH 5.5 containing 0.5M NaCl. The resulting sample was then desalted by gel filtration through a G25 column (Pharmacia) equilibrated at 1X with PBS to a 95% purity level. Protein concentrations were determined by colorimetric methods as described by Lowry et al (1951) J.biol.chem.191, 495-498. The fusion protein was characterized by western blotting techniques using antibodies specific for P64K and TGF α.

Example 6: the recombinant protein TGF alpha-P64K was recognized by hTGGF alpha specific monoclonal antibodies (mabs).

Western blotting techniques were used to determine whether TGF α in the fusion protein could be recognized by anti-hTGF α mab (calbiochem). 25 μ g of EGF-P64K, TGF α -P64K or P64K were electrophoretically separated in two polyacrylamide gels and then transferred to 0.45 μm nitrocellulose membranes according to conventional procedures. After transfer, the membranes were incubated overnight at 4 ℃ with TBS 1 × blocking solution containing 5% skim milk. After simple washing with TBS 1 × -Tween20 (0.05%), one membrane was incubated with anti-P64K antibody (1/500) (fig. 3A) and the other membrane with anti-TGF α Mab (1/100) (fig. 3B) for 2 hours at room temperature. The membrane was then washed 3 times with the same solution and incubated with alkaline phosphatase-labeled goat anti-mouse immunoglobulin (1/1000) for 1 hour under the same conditions. Finally, 0.004g of Fast Net enzyme substrate (Sigma) in 20mL buffer containing 0.1M Tris-Cl (pH 8.2), 0.004g naphthol ACE-MX phosphate (Sigma) and 400 μ L NN' dimethylformamide was added. The reaction was stopped with a similar rinse. Specific recognition of TGF α -P64K by anti-hTGF α mabs was observed (figure 3). This result demonstrates that TGF α in the fusion protein retains a structure that can be recognized by specific antibodies.

Example 7: the chemical conjugate hTGFa-P64K was obtained.

1ml was added in PBS/10mM MgCl₂TGF alpha at a medium concentration of 2mg/mL was mixed with 1mL of P64K at a concentration of 2mg/mL in the same solvent. Then 0.2ml of 0.5% glutaraldehyde solution was added to make the final percentage 0.05%. The mixture was incubated at room temperature for 1 hour, then 1X/10 mM MgCl in PBS₂And (5) dialyzing the solution. Finally, the mixture was dialyzed at 4 ℃ against PBS 1X overnight. The immunogenic formulation is filtered under sterile conditions and stored at 4 ℃ until use.

Example 8: obtaining the fusion protein of hTGFa, hEGF and P64K.

The gene encoding hEGF (150bp) was PCR amplified using plasmid pBEF 10 as template. This plasmid contains the entire hEGF cloned into the EcoR V site of the commercial vector pBluescript SKII (Stragene). The obtained DNA was ligated to the BamHI site of pMHisTGF α plasmid at the C-terminus of P64K using the method described in example 2. Thus, the pMTGF-EGF vector encoding the fusion protein TE-P64K was obtained.

Example 9: the chemical binder hTGF α -hEGF-P64K was obtained.

1ml was added in PBS/10mM MgCl₂TGF alpha at a medium concentration of 3mg/mL was mixed with 1mL of hEGF at a concentration of 3mg/mL in the same solvent and 3mg/mL of P64K. Then 0.6mL of 0.5% glutaraldehyde solution was added to make the final percentage 0.05%. The mixture was incubated at room temperature for 1 hour, then 1X/10 mM MgCl in PBS₂And (5) dialyzing the solution. Finally, the mixture was dialyzed at 4 ℃ against PBS 1X overnight. Filtering the mixture under aseptic conditionsImmunogenic formulations and stored at 4 ℃ until use.

Example 10: preparation of hTGF alpha-containing formulation.

The different immunogenic formulations described in examples 2,3, 7, 8 and 9 were combined with Al (OH) as detailed in the present invention₃Or Montanide ISA 51. The amount of hTGFa in all formulations is equal to 50 μ g and in the combination vaccines described in examples 8 and 9 the amount of hTGFa and hEGF is 50 μ g. 2mg of Al (OH) was used for each fusion protein or chemical conjugate preparation₃Formulations contain 50 μ g of hTGFa or hEGF equivalent, respectively.

Example 11: preparing the combined vaccine containing the TGF alpha-P64K protein and the EGF-P64K protein.

Prior to injection, 50 μ g of each growth factor in a total volume of 0.5mL in 0.6mg of recombinant was mixed with the same volume of Montanide ISA 51 and shaken for 10 minutes at room temperature.

When Al (OH) is used₃As an adjuvant, 2mg Al (OH) will be injected before₃The two preparations containing 0.6mg of each protein were mixed.

Example 12: preparing a combined vaccine containing the chemical combination of TGF alpha/P64K and EGF/P64K.

0.25mL of an immunogenic formulation containing 50 μ g of hTGF α conjugated to P64K as described in example 7 was mixed with 0.25mL of the corresponding immunogenic formulation containing hEGF using a syringe at room temperature and mixed with 0.5mL of Montanide ISA 51 for 10 minutes as described in example 10.

When Al (OH) is used₃When used as an adjuvant, the solution is adsorbed onto 2mg of Al (OH)₃0.5mL of each of the above chemical binders containing 50. mu.g of hTGFa or hEGF, respectively, was mixed.

Example 13: immunogenicity of TGF-P64K/incomplete Freund's adjuvant (MontanideeISA 51) in a mouse model.

To confirm the immunogenicity of the vaccine, 6-8 week old female Balb/c mice were injected subcutaneously with 58mg (equivalent to 5. mu.g TGF. alpha.), 116mg (10. mu.g) or 0, 6mg (50. mu.g) TGF. alpha. -P64K mixed 1: 1 with Montanide ISA 51. Immunogens were prepared and shaken for 10 minutes prior to immunization as detailed in the present invention. Each animal was inoculated with 4 doses. Blood was drawn before, one week after and every two weeks thereafter. Serum was isolated from the blood of the drawn animal and the specific antibody titer against hTGF α was determined using an indirect ELISA technique.

Briefly, microtiter ELISA plates (COSTAR) were coated with 50 μ L/well of pH 7.2 carbonate-bicarbonate buffer containing 2.5 μ g/mL hTGF α and incubated overnight at 4 ℃. After three washes with PBS 1X-Tween 20 (0.05%), it was blocked with PBS 1X-Tween 20 (0.05%) -SFT (5%) solution at 37 ℃ for 1 hour. Serum from immunized mice was immediately added and incubated at 37 ℃ for 2 hours. After washing, alkaline phosphatase-labeled goat anti-mouse immunoglobulin (Sigma) diluted 1/1000 in PBS 1X-Tween 20 (0.05%) -SFT (5%) was added to the plate and the plate was incubated at the same temperature for 1 hour (50. mu.L/pozo). Finally, after washing, the enzyme substrate (p-nitrophenyl phosphate (Sigma)) (50 μ L/well) was added at a final concentration of 1mg/mL in diethanolamine buffer, pH 9.8. The absorbance of the enzyme-substrate complex formed at 405nm was measured in an ELISA plate reader.

FIG. 4 shows the kinetics of multivalent anti-hTGFa antibody responses obtained in mice immunized with TGF-P64K.

Due to the high homology (93%) between hTGFa and the corresponding rat or mouse factor, this immune response against hTGFa can be considered as a response against autologous TGF alpha (mouse).

Example 14: TGF alpha-P64K/Al (OH) in mouse model₃The immunogenicity of (a).

2mg of Al (OH) are used₃As an adjuvant, immunization protocols were performed as described in the previous examples. The immunogenic formulations were prepared as described in detail in the present invention. The antibody titer obtained for TGF α was 1/10000. The technique used to determine antibody titers was the indirect ELISA described in example 13.

Example 15: distribution of IgG subpopulations in mice immunized with TGF α -P64K protein/incomplete freund's adjuvant (Montanide ISA 51) in a mouse model.

The distribution of IgG subpopulations was determined by indirect ELISA technique as described in example 13 using 1/1000 diluted specific antisera coupled to biotin (Jackson) against different IgG subpopulations, followed by streptavidin-phosphatase complex (1/1000).

The proportion of each IgG subpopulation relative to total IgG in the serum of the immunized animal was determined. Animals were immunized with fusion proteins containing 50 μ g of TGF α by subcutaneous (group 1) or intramuscular (group 2) routes following the immunization procedure described in example 13. Figure 5 is the obtained subpopulation distribution. Both groups of mice used in the study received a greater proportion of IgG 1.

Example 16: measurement of inhibition of serum I by radioreceptor assay technique (RRA) in animals immunized with TGF-P64K protein/incomplete Freund's adjuvant (Montanide ISA 51)¹²⁵-the ability of TGF-alpha to bind.

To determine whether the antibodies generated during the immunization program described previously are capable of inhibiting TGF-alpha binding to its receptor, an in vitro technique known as RRA was used. In synthesis, sera from immunized mice obtained as described in example 13 were combined with serum containing 100mL human placental membrane and 20mLI¹²⁵-TGF α (100000cpm) and 330mL buffer (pH 7.4, 10mM Tris-Cl, 10mM MgCl₂And 1% BSA). Coupling of TGF-alpha to the radioisotope I Using the chloramine-T method (Hunter and Greenwood (1962), Nature, 358: 495-498)¹²⁵. The mixture was incubated at room temperature for 1 hour and the reaction was stopped with 1mL of the previously mentioned buffer. Finally the tubes were centrifuged at 1000rpm for 30 minutes. The precipitate was washed and dried. Radioactivity was detected with a gamma emission counter (Wallac, Finland). A decrease in the radioactivity measurement indicates that binding of TGF α to its receptor is inhibited by the action of the serum being tested. The percent inhibition was 50% -80% in all sera tested.

Example 17: the humoral response against human EGF (hEGF) generated by immunization with TGF-P64K protein was determined.

The presence of anti-EGF antibodies was detected in mouse sera showing high anti-TGF-alpha antibody titers using the indirect ELISA technique described. To plates coated with hegf (cigb) were added 1/100, 1/1000 and 1/10000 diluted sera. FIG. 6 shows the anti-EGF antibody titers obtained in the sera of mice immunized with TGF-P64K protein. Positive anti-EGF antibody responses were obtained in only one group of immunized mice.

However mice immunized with one of the chemical conjugates EGF-P64K did not show any level of anti-TGF-alpha antibodies.

Example 18: polyclonal antiserum anti-hTGF alpha obtained by immunization with TGF alpha-P64K protein/incomplete Freund's adjuvant (MontanideeISA 51) identifies human tumors in vitro.

Polyclonal antiserum anti-hTGFa obtained by immunizing mice with TGF alpha-P64K protein and Montanide ISA 51 was used to detect TGF alpha expression in tumor biopsies embedded in paraffin. These biopsies were obtained from patients vaccinated with EGF based vaccines. Regression of NSCLC tumors was observed in one patient with a high anti-hEGF antibody response. However, a second tumor of the larynx was later detected. Biopsies of both tumors were analyzed and differential expression of EGF and TGF α in each tumor was observed. The reactivity values for the different antibodies are shown in FIG. 7. These results confirm the fact that immunization with a vaccine formulation containing TGF-P64K will generate anti-hTGFa-specific antibodies that recognize hTGFa in human tumors.

Example 19: mRNA expression of EGF, TGF alpha and EGF-R in breast cancer biopsies.

Messenger ribonucleic acid (mRNA) was isolated from breast cancer tumor biopsies using TRIZOL reagent (Life technologies) and converted to cDNA by reverse transcriptase. Total cDNA was subjected to 30 PCR cycles using specific primers for each of these molecules. A housekeeping Gene (GAPDH) was used as an internal control. The obtained PCR products were electrophoretically separated on a 1.5% agarose gel and visualized with ethidium bromide.

FIG. 8 shows the results obtained in 22 breast cancers using specific primers for EGF, TGF α, EGF-R and GAPDH (internal control). EGF mRNA was observed only in the biopsy of 1/22, while high expression of TGF alpha and EGF-RmRNA was observed in most samples. The high correlation between the expression of these two molecules suggests the importance of the TGF-alpha/EGF-R autocrine loop in the growth of this type of tumor (FIG. 9).

Brief description of the drawings

FIG. 1: the gene and amino acid sequence of mature hTGFa (bold underlined letters).

FIG. 2: schematic representation of the process for obtaining the expression vector pMTGF alpha.

FIG. 3: recognition of the TGF alpha-P64K fusion protein by Western blotting techniques, anti-P64K mab (A) and anti-hTGFalpha mab (B). 10% SDS-PAGE was performed for P64K (1), EGF-P64K (2) and TGF α -P64K (3). The proteins were then transferred to nitrocellulose membranes and incubated with specific antibodies against P64K (A) or TGF α (B) to characterize fusion proteins of TGF α and P64K.

FIG. 4: anti-hTGF α antibody reaction kinetics: the titer of anti-hTGF α specific antibody was determined by indirect ELISA technique. Mice were immunized with TGF-P64K protein equivalent to 5. mu.g (A), 10. mu.g (B), and 50. mu.g (C) hTGFa mixed with Montanide ISA 51. The x-axis represents the number of days a sample was collected from each mouse and the y-axis represents the reciprocal of the antibody titer achieved. The days of immunization are indicated by arrows in panel a (days 0, 14, 28 and 42).

FIG. 5: distribution of IgG subpopulations induced by immunization with fusion proteins equivalent to 50. mu.g of TGF-alpha. Comparison of the proportion of IgG subpopulations in antibody responses induced by subcutaneous (1) or intramuscular (2) immunization with TGF-alpha-P64K protein. Standard deviation values are shown for 5 immunized animals per group.

FIG. 6: anti-EGF specific antibody response in mice immunized with TGF-alpha P64K fusion protein. Shown in the graph are titers of anti-hTGFa and anti-EGF antibodies achieved in mice immunized with TGF alpha-P64K.

FIG. 7: expression of EGF-R, EGF and TGF alpha was determined by immunohistochemical analysis of tumor biopsies from vaccinated patients in a phase II clinical trial of EGF vaccine. The differential activity of these three molecules in primary lung tumors and secondary laryngeal primary tumors occurs later, as shown by the plus sign in the figure.

FIG. 8: expression of EGF, hTGFa and EGF-R mRNA in 22 breast cancers. The figure shows the products of 30 PCR cycles observed with ethidium bromide after being obtained with primers specific for each molecule and separated on a 1.5% agarose gel. GAPDH mRNA expression was also seen as an internal control.

FIG. 9: relationship between hTGFa and EGF-R mRNA levels in breast cancer biopsies: the intensity of the band obtained with ethidium bromide was analyzed by a calculation program (ImagQuant, Amersham). The x-axis shows the relationship (relative intensity) between the intensity values of the PCR product obtained with primers specific for EGF-R and the intensity values obtained with GAPDH for each sample, and the y-axis is the same relative intensity value for hTGFa. A positive correlation was observed between the expression of these two molecules (R)²＝0.657，P＝0.00121)。

Claims (14)

1. Vaccine composition for eliciting a specific immune response against auto-TGF α, wherein said composition comprises auto-TGF α or any derivative thereof as active ingredient together with a suitable adjuvant.

2. The vaccine composition according to claim 1, comprising human recombinant TGF α.

3. Vaccine composition according to claims 1 and 2, comprising as active ingredient self TGF α alone or in combination with other EGF-R ligands.

4. A vaccine composition according to claims 1-3, wherein self TGF α and EGF-R ligands as active ingredients may optionally be coupled to the carrier protein by chemical binding or by genetic means.

5. A vaccine composition according to claim 4, which comprises the P64K protein from Neisseria meningitidis as a carrier protein.

6. A vaccine composition according to claim 5, comprising as active ingredient a chemical conjugate between TGF α and the P64K protein from Neisseria meningitidis.

7. A vaccine composition according to claim 5, which comprises as active ingredients a chemical conjugate between TGF α and the P64K protein from Neisseria meningitidis, and further comprises EGF.

8. A vaccine composition according to claim 5, which comprises as active ingredient a recombinant fusion protein between TGF α and the P64K protein from Neisseria meningitidis.

9. A vaccine composition according to claim 5, comprising as active ingredient a recombinant fusion protein between TGF α and the P64K protein from Neisseria meningitidis, wherein the N-terminus of the P64K protein has a six histidine tail.

10. A vaccine composition according to claim 9, comprising as active ingredients a recombinant fusion protein between TGF α, the P64K protein from neisseria meningitidis and EGF.

11. A vaccine composition according to claims 1-10, comprising incomplete freund's adjuvant as adjuvant.

12. Vaccine composition according to claims 1-10, comprising al (oh)₃As an adjuvant.

13. A method of treating a malignant disease of a tumour of epithelial origin expressing TGF α or any EGF-R ligand using a vaccine composition according to any one of claims 1 to 12.

14. A method according to claim 13 for the treatment of tumors, such as epidermoid carcinoma of the lung, breast, prostate, stomach and ovary, wherein a vaccine composition according to any one of claims 1-12 is used.