DIVALENT VACCINE COMPOSITIONS AND THE USE THEREOF FOR TREATING TUMORS

Abstract

The invention describes vaccine compositions combined in the same proportion with the extracellular domains of growth factor receptors Her1 and Her2 or fragments thereof and furthermore very small size proteoliposomes derived from proteins of the outer membrane of Neisseria meningitidis and GM3 ganglioside (VSSP-GM3), administered subcutaneously. The disclosed compositions, which induce the production of antibodies are used for the treatment of malignancies and offer advantages because they completely remove the tumor mass thus preventing tumor regression due to the emergence of resistant variants.

Claims

1-8. (canceled)

8. The vaccine composition of claim 1 for use in inducing an immune response in a treatment administered subcutaneously every two weeks to complete a total of 5 doses and subsequent monthly as maintenance dose for at least one year.

9. A method for the treatment of tumors that co-express Her1 and Her2, said method comprising the step of administering to a subject in need thereof a vaccine composition comprising the ECDs of Her1 and Her2 receptors or fragments thereof in the same proportion and concentration sufficient to produce an immune response against malignant tumors that express Her1 and Her2 receptors, in combination with very small size proteoliposomes derived from proteins of the outer membrane of Neisseria meningitides and GM3 (VSSP-GM3) in, wherein said ECDs of Her1 and Her2 receptors are in the same proportions and are each in a concentration of from 100 to 800 g/dose.

10. The method for claim 9, wherein said composition further comprises a pharmaceutically acceptable adjuvant.

11. The method of claim 10, wherein the pharmaceutically suitable adjuvant is Montanide ISA 51.

12. The method of claim 10, wherein the pharmaceutically suitable adjuvant is Alumina.

13. The method of claim 9, where the vaccine composition is administered subcutaneously.

14. The method of claim 8 wherein the vaccine composition is administered subcutaneously every two weeks to complete a total of 5 doses and subsequently monthly as maintenance dose for at least one year.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG. 1 shows the kinetics of IgG Abs in BALB/c mice (n=5) immunized subcutaneously four times every 14 days, and 30 days after the last immunization with 100 g of ECD-Her1 and 100 g of ECD-Her2 in 200 g of VSSPs. Blood draws were performed on days 7, 21, 35, 56, 87 and 102 to measure IgG Abs titers in the immune sera using ELISA assay. Abs IgG kinetics are expressed as the Log (1/titre media+1) for each extraction day, when it was coated with the ECD-Her1 (a) and ECD-Her2 (b). Abs response of individual mice (n=5) is shown in the figure, the response is specific to ECD-HER1 (c) and ECD-Her2 (d) and was measured by plotting the reciprocal logarithm of the Abs title plus 1, on day 87 of the immunization protocol. Different letters indicate statistical significance as tested by two-way ANOVA (treatment group factor), using Bonferroni test correction (p<0.05).

[0028] FIG. 2 shows the recognition of MDAMB468 and MCF7/HER2 tumor cell lines by immune sera induced by Bivalent Vaccine Her1+Her2 and monovalent vaccines Her1 and Her2, using flow cytometry. The aforementioned human tumor cell lines were incubated with the immune sera (n=5) or with a mixture of pre-immune sera diluted 1:200, in day 35 of the immunization protocol, as well as the controls of the expression of receptors: MAbs nimotuzumab and trastuzumab, at a concentration of 10 g/ml. Subsequently, the cells were labeled with goat PAbs anti-mouse IgG conjugated with fluorescein isothiocyanate. The labeled cells were visualized by flow cytometry. The percentage of cells recognized by the sera and the MAbs was determined using Flow Jo software version 5.7.2. Different letters indicate statistical significance as tested by two-way ANOVA (treatment group factor), using Bonferroni test correction (p<0.05).

[0029] FIG. 3 shows the recognition of H292 tumor cell line by immune sera induced by Bivalent Vaccine Her1+Her2 and monovalent vaccines Her1 and Her2, using flow cytometry. Human tumor cells were incubated on day 35 of the immunization protocol with immune sera (n=5) or with a mixture of pre-immune sera diluted 1:300, as well as the controls of the expression of receptors: MAbs nimotuzumab and trastuzumab at concentration of 10 g/ml. Subsequently, the cells were labeled with goat PAbS anti-mouse IgG conjugated with fluorescein isothiocyanate. The labeled cells were visualized by flow cytometry. The percentage of cells recognized by the sera and MAbs was determined using Flow Jo software version 5.7.2. Different letters indicate statistical significance as tested by two-way ANOVA (treatment group factor), using Bonferroni test correction (p<0.05).

[0030] FIG. 4 shows the inhibition of Her1 and Her2 phosphorylation caused by the immune sera induced by Bivalent Vaccine Her1+Her2 administration. H292 cells were incubated in a mixture of five immune sera induced by Bivalent Vaccine Her1+Her2 and Monovalent Vaccines Her1 and Her2. Levels of Her1 and Her2 phosphorylation after stimulation with EGF and with the different treatments were determined by Western blot. Untreated cells were used as negative control and the treatment with a mixture of pre-immune sera (PI) as a negative control of specificity. TKI AG1478 was used as positive control for inhibition. The pictures show the levels of P-Her1 (a) and P-Her2 (b) obtained with different treatments. Bar graphs show the densitometric analysis of the pictures from one representative experiment chosen from the two experiments performed.

[0031] FIG. 5 shows the anti-proliferative effect of the Abs induced by Her1+Her2 bivalent vaccine on H292 tumor line. H292 cells were incubated for 48 hours in a mixture of five immune sera, diluted 1:20, on day 87 of the immunization protocol. The mixture of PI sera was used as negative control in the experiment. Cell viability was determined by the MTT colorimetric method, determining the difference of optical density (OD) by subtracting OD at 540 to the value of OD at 620 nm. The maximum viability percentage was found when subtracting the difference of absorbance of the incubation with PI serum. Bars represent the mean of triplicates of three experiments for each treatment. Different letters indicate statistical significance according to Dunnett T3 test for a vs b, p<0.01 for a vs c, p<0.001.

[0032] FIG. 6 shows the titers of Abs that recognize ECD-Her1 and ECD-Her2 induced by Bivalent Vaccine Her1+Her2 formulations that contain equal or not equal amounts of each receptor. BALB/c mice (n=5) were immunized subcutaneously four times every 14 days with: Group 1, 100 g of ECD-Her1 and 100 g of ECD-Her2; Group 2, 100 g of ECD-Her1 and 200 g of ECD-Her2; Group 3, 100 g of ECD-Her1 and 300 g of ECD-Her2; Group 4, 200 g of the ECD-Her1 and 100 g ECD-Her2; Group 5, 300 g ECD-Her1 and 100 g ECD-Her2. All formulations contained 200 g of VSSP. The determination of ECD-Her1 and ECD-Her2 specific antibody titers was performed on the serum by means of an ELISA assay, from a blood draw taken on day 56. Abs IgG kinetics are expressed as the media of log reciprocal plus one for each extraction day, when it was coated with the ECD-Her1 (a) and ECD-Her2 (b). IgG Abs response of individual mice (n=5) was determined by plotting the inverse of the antibody titers.

[0033] No statistical differences as tested by two-way ANOVA (treatment group factor), using

[0034] Bonferroni test correction (p<0.05) were observed. The following examples are meant only to illustrate, but in no way to limit, the claimed invention.

EXAMPLES

Example 1: Specific Antibodies Titles Raised Against ECD-Her1 and DEC-Her2 Induced by Her1+Her2 Bivalent Vaccine

[0035] Balb/c mice were immunized subcutaneously with a bivalent vaccine formulation containing 100 g of ECD-Her1 and 100 g of ECD-Her2. A second group of mice was immunized with a monovalent vaccine formulation containing 100 g of ECD-Her1, and a third group was immunized with a monovalent vaccine formulation containing 100 g of ECD-Her2. The three vaccine formulations mentioned contained 200 g of VSSP adjuvant. Immunizations were performed on days 0, 14, 28, 42 and 72, and blood was drawn for processing the serum at days 2, 21, 56, 87 and 102. Specific antibody titers against the ECDs of Her1 and Her2 in the serum were determined using ELISA method.

[0036] The mice immunized with Her1+Her2 Bivalent vaccine raised specific IgG isotype antibodies against the ECD-Her1 and the ECD-Her2. The antibody titers induced against each of these receptors did not differ from those induced by the respective monovalent vaccines. In the case of the antibody response against ECD-Her1, both the titers induced by Her1+Her2 bivalent vaccine and the monovalent vaccine reached 1/10.000 values. The response against ECD-Her2 induced both by the bivalent and the monovalent vaccine reached antibody titers values of 1/200.000 (FIG. 1).

[0037] This result shows that mixing the two receptors in a single vaccine formulation does not affect the immunogenicity of each of them individually, which validates the potential use of this vaccine formulation.

Example 2: Recognition of Her1+/Her2.SUP.., Her17Her2+Tumor Lines by Sera Induced by Bivalent Vaccine Her1+Her2

[0038] The sera from mice immunized with the Bivalent Vaccine Her1+Her2 and Monovalent Vaccines Her1 and Her2, diluted 1/200 were incubated with 10.sup.5 cells of different tumor cell lines: MDA-MB468 (ATCC, HTB-132), derived from breast adenocarcinoma, and MCF7/Her2, generated from the transfection of MCF7 (ATCC HTB-22) cell line with full-length Her2. The PI sera were used as negative specificity controls. The Mab nimotuzumab, that recognizes Her1, and Herceptin monoclonal antibody, that recognizes Her2, were used as positive controls.

[0039] The sera generated by the Bivalent Vaccine recognized the same percentage of MDA-MB468 cells as the sera generated by Monovalent Vaccine Her1. Furthermore, the sera generated by the Bivalent Vaccine recognized the same percentage of MCF7/Her2 cells as the sera generated by Monovalent Vaccine Her2 according to Dunnett's T3 (p>0.05) test (FIG. 2). The intensity of recognition of these receptors was also very similar (Table 1).

[0040] This demonstrates that the antibodies induced the bivalent formulation containing the truncated Her1 and Her2 receptors does not affect the recognition of the full-size Her1 and Her2 receptors in the membrane of tumor cells. It also shows that the quality of this recognition is not affected when the antibodies against Her1 and Her2 are generated from a formulation based on both receptors, since the recognition was the same as that obtained with the sera from the monovalent vaccines both in number of recognized cells and the intensity of the recognition.

TABLE-US-00001 TABLE 1 Mean fluorescence intensity (MFI) average of MDAMB468 and MCF7/HER2 cells recognized by the immune sera generated by the treatments. MDAMB468 MCF7/HER2 Treatments MFI MFI Vac Her1 .Math. Her2 410.05 140.99 Vac Her1 370.23 4.31 Vac Her2 4.02 104.05

Example 3: Recognition of Her1+/Her2+Tumor Line by Sera Induced by Her1+Her2 Bivalent Vaccine

[0041] Sera from mice immunized with Her1+Her2 Bivalent Vaccine and monovalent vaccines Her1 and Her2, diluted 1/300 were incubated with tumor cell line H292 (ATCC CRL-1848), derived from squamous cell carcinoma of the lung. The pre-immune sera were used as negative specificity controls. MAb nimotuzumab and MAb Herceptin were used as positive controls.

[0042] The percentage of H292 cells recognized was statistically higher in the sera generated by the bivalent vaccine, as compared to the recognition of the sera generated by Her1 and Her2 monovalent vaccines, according to Dunnett T3 test (p<0.05) (FIG. 3).

[0043] This demonstrates that the polyclonal antibodies induced by the bivalent formulation have a higher threshold of recognition of tumor cells due to its ability to simultaneously recognize both receptors, Her1 and Her2, on the membrane of cells. This suggests that the bivalent vaccine has a higher potential with respect to the monovalent vaccines, in terms of biological effect on H292 tumor cells, determined by the number of recognized receptors. Such is the case of the inhibition of activation of the receptors from the Her family, which are involved in the proliferation of tumors.

Example 4: Inhibition of the Activation of HER1 and HER2 Receptors by Bivalent Her1+Her2 Vaccine

[0044] Sera from mice immunized with Bivalent Vaccine Her1+Her2 and monovalent vaccines Her1 and Her2, diluted 1/100 were incubated with cells from H292 tumor cell line. The cells were stimulated with 100 ng/mL of EGF for 10 min and then lysed. The effect of immune sera on the inhibition of EGFR phosphorylation was determined by Western blotting assay, using specific antibodies for the detection of phosphorylated EGFR and total EGFR. In this assay AG1478 tyrosine kinase inhibitor at 10 M was used as positive control of the phosphorylation inhibition, and PI serum was used as negative control of specificity.

[0045] Sera generated by the Bivalent Vaccine inhibited the activation of Her1 and Her2 receptors, measured in terms of phosphorylation, to a greater extent than sera of monovalent vaccines Her1 and Her2. The films from western blotting were subjected to densitometric analysis. The data from the densitometric analysis yielded that the decrease in Her1 phosphorylation caused by the immune sera induced by Bivalent Vaccine Her1+Her2 with respect to PI serum was 11.2 times. When the same analysis was performed to the sera induced by monovalent vaccine Her1, the decrease of Her1 phosphorylation was 1.7 times. In the case of sera from the Monovalent Vaccine Her2 group the decrease was 2.2 times. In the case of Her2, the decrease of Her2 phosphorylation induced by sera from mice immunized with Her1+Her2 Bivalent Vaccine with respect to PI serum was 8.7 times. When the same analysis was performed to the sera induced by monovalent vaccine Her1, the decrease in Her2 phosphorylation was 1.7 times, and for the sera from the Monovalent Vaccine Her2 group the decrease was 3.5 times (FIG. 4).

[0046] This demonstrates the superiority of the Bivalent Vaccine with respect to the Monovalent Vaccines. The Bivalent Vaccine could not only be able to directly inhibit the phosphorylation of Her1 or Her2 receptors, that form homodimers (Her1/Her1 and Her2/Her2) through antibodies that block ligand binding and against the dimerization arm, but it could also be more efficient than the monovalent vaccines in reducing the activation of the receptors that are part of a Her1/Her2 heterodimer.

Example 5: Anti-Proliferative Effect of the Sera Induced by Her1+Her2 Bivalent Vaccine on Her1+/Her2+Tumor Line

[0047] H292 cells were incubated for 48 h with sera from mice immunized with Bivalent Vaccine Her1+Her2 and Monovalent Vaccines Her1 and Her2, diluted 1:20. After this time, the culture supernatant was removed and the MTT reagent was added at a concentration of 1 mg/mL. After 4 hrs of incubation, the supernatant was removed and 100 L of dimethylsulf oxide were added to dissolve the formazan crystals; absorbance was measured determining the difference of OD by subtracting OD at 540 to the value of OD at 620 nm with an ELISA reader. Viable cells were evaluated by determining the difference in absorbance between 540 nm and 620 nm. The difference of absorbance obtained in the PI serum was taken as maximum viability percentage.

[0048] Tumor cells treated with the sera induced by Bivalent Vaccine Her1+Her2 had less viability than tumor cells incubated with the serum induced by the monovalent vaccines, according to Dunnett's T3 statistic test (p<0.05 (FIG. 5). This demonstrates that the administration of the Bivalent Vaccine induced a higher antitumor effect on cells, in terms of anti-proliferative effects, than the monovalent vaccines expressing Her1 and Her2 receptors. This suggests its potential to be an effective therapeutic agent against epithelial tumors expressing both receptors.

Example 6: Non-Equivalent Doses of ECD-Her1 and ECD-Her2 Decrease the Capacity of the Antibodies Generated to Recognize H125/Her1+/Her2+

[0049] Balb/c mice were immunized subcutaneously with a bivalent vaccine formulation containing non-equivalent combinations of the ECD-Her1 and ECD-Her2. Group 1 was immunized with 100 g of ECD-Her1 and 100 g ECD-Her2; group 2 with 100 g of ECD-Her1 and 200 g ECD-Her2; group 3 with 100 g of ECD-Her1 and 300 g of ECD-Her2; group 4 with 200 g of ECD-Her1 and 100 g of ECD-Her2; group 5 with 300 g of ECD-Her1 and 100 g ECD-Her2. All mentioned adjuvant vaccine formulations contained 200 g of VSSP. Immunizations were given on days 0, 14 and 28 via subcutaneous injections and blood was drawn to process the serum on day 42. Specific antibody titers against the ECDs of Her1 and Her2 were determined using ELISA method and the capacity of the above-mentioned sera to recognize H125 tumor cell line, which expresses Her1 and Her2, was assessed by FACS (FIG. 6).

[0050] The percentage of tumor cells recognized by the immune sera was 100% for all groups evaluated, which is explained by the ability of formulation variants evaluated to induce antibody titers against Her1 and Her2. However, the recognition quality, measured in terms of mean fluorescence intensity (MFI) was higher for group 1, wherein equivalent doses of both receptors (Table 2) were combined.

TABLE-US-00002 TABLE 2 Recognition of H125 tumor cell line by the immune sera induced by Her1 + Her2 bivalent vaccine formulations containing equivalent and non-equivalent doses of each receptor. Groups G1 G2 G3 G4 G5 ECD-Her1 (g) 100 100 100 200 300 ECD-Her2 (g) 100 200 300 100 100 Recognized cells (%) 100 100 100 100 100 MFI 90.86 71.3 45.7 60.6 53.63 MFI: mean fluorescence intensity