METHOD FOR THE TREATMENT OF PATIENTS WITH CARCINOMAS

Abstract

The present invention relates to the branch of Biotechnology and Medicine, particularly to a method for the selection and treatment of patients with carcinomas of epithelial origin that co-express the HER1 and HER2 receptors without increased expression of these receptors or with the presence of RAS activating mutations. In particular, this method is based on the application of bivalent vaccine compositions which have as active principle the extracellular domains of the HER1 and HER2 receptors or portions thereof and as an adjuvant the very small proteoliposomes derived from the outer membrane proteins of Neisseria meningitidis and the GM3 ganglioside. This method is useful for the treatment of patients whose expression levels of HER1 and HER2 do not allow the monoclonal antibodies against HER1 and/or HER2 to have a therapeutic effect.

Claims

1. A method for determining whether a patient diagnosed with carcinoma of epithelial origin is likely to respond to treatment with an inhibitor of the epidermal growth factor receptor (EGFR) family which comprises a step of determination in a sample taken from said patient the presence of at least one of the following biomarkers: co-expression of the HER1 and HER2 receptors and their expression levels, activating mutations in the RAS family genes.

2. The method according to claim 1 wherein it is concluded that the patient is likely to respond to the treatment if: there is co-expression of the HER1 and HER2 receptors without increased expression of them or, there is presence of activating mutations in the RAS family genes.

3. The method according to claim 2 wherein patients are considered responders if the level of expression of HER1 and HER2 in the sample taken from them has a score of 1+ or 2+ measured by immunohistochemistry.

4. A method for treating a patient with carcinoma of epithelial origin comprising: the determination by means of the method according to claim 1 whether a patient with carcinoma of epithelial origin is likely to respond to treatment with an inhibitor of the EGFR family or not and the administration to patients who are likely to respond to treatment as determined in a) of a therapeutically effective amount of an EGFR family inhibitor.

5. The method according to claim 4 wherein the EGFR family inhibitor is the bivalent vaccine (BV) which has as its active ingredient the extracellular domains of the HER1 and HER2 receptors or portions thereof and as adjuvant the very small size proteoliposomes derived from the outer membrane proteins of Neisseria meningitidis and GM3 ganglioside.

6. The method according to claim 5 wherein the dose range of the extracellular domains of HER1 and HER2 or portions thereof is between 400 g to 1600 g; being this concentration the sum of both molecules.

7. The method according to claim 5 wherein the BV is administered subcutaneously, intradermally or intramuscularly with weekly during the first five doses followed by monthly doses of maintenance by at least six months by subcutaneous, intradermal or intramuscular route.

8. The method according to claim 4 for the treatment of squamous epithelial cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon and rectum carcinoma, head and neck cancer, uterus cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulva cancer, thyroid cancer, anal carcinoma and carcinoma of the penis.

9. The method according to claim 4 wherein the responder patients show a complete or partial response after the administration of 9 doses of the BV.

10. The use of the method according to claim 1 for the treatment of carcinomas of epithelial origin.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0041] FIG. 1. Recognition by flow cytometry of tumor lines with different expression levels of HER1 and HER2: A431, MDAMB468, H125, H292, PC3, SKBR3, MCF7/HER2, SKOV3, A549 and Raji, by polyclonal antibodies (PAbs) generated by the HER1+HER2 BV.

[0042] FIG. 2. Effect caused by the PAbs induced by the HER1+HER2 BV on cell viability: A) Flow cytometry performed to evaluate the recognition of the lines tested by MAbs trastuzumab and nimotuzumab. B) Inhibition of cell viability in cell lines A431, H125, H292, SKOV3, SKBR3 with differential expression of HER1 and HER2 measured by the colorimetric method MTT. C) Comparative bar graph of the effect of sera on cell viability in the different lines tested.

[0043] FIG. 3. Inhibition measured by Western Blot in the H292 tumor line of the activation of HER1 and HER2 receptors and signaling cascade proteins, caused by the PAbs of the immune serum induced by the HER1+HER2 BV. PI: Pre-immune serum, TKI: tyrosine kinase inhibitor.

[0044] FIG. 4. Effect measured by Western Blot of the PAbs induced by the HER1+HER2 BV on the degradation of the HER1 and HER2 receptors in the H292 tumor line.

[0045] FIG. 5. Comparison of the effect induced by the PAbs generated by the HER1+HER2 BV, the MAbs nimotuzumab and trastuzumab and the combination of both, on the cell viability of the lung carcinoma line H292.

[0046] FIG. 6. Comparison measured by Western Blot of the capacity of the PAbs induced by the HER1+HER2 BV and the MAbs nimotuzumab, cetuximab and trastuzumab to inhibit the activation of the HER1 and HER2 receptors in the tumor lines H125 and H292.

[0047] FIG. 7. Comparison measured by ELISA of the capacity of the PAbs induced by BV with the MAbs nimotuzumab, cetuximab and trastuzumab to degrade the HER1 and HER2 receptors in the H292 tumor line. A) Degradation of HER1 after 24 hours of treatment. B) Degradation of HER2 after 1 h of treatment.

[0048] FIG. 8. Comparison of the effect of the PAbs induced by the HER1+HER2 BV on the viability of the KRAS mutated lung carcinoma A549 cell line with the effect of the MAbs nimotuzumab, cetuximab, trastuzumab, and its combinations.

[0049] FIG. 9. Comparison of the capacity of the PAbs induced by the HER1+HER2 BV with that of the MAbs nimotuzumab, cetuximab, trastuzumab, and its combinations to degrade the HER1 and HER2 receptors in the KRAS mutated lung carcinoma A549 cell line

EXAMPLES

Example 1. Recognition of Tumor Lines HER1+HER2+ by the PAbs of the Immune Serum Induced by the HER1+HER2 BV

[0050] Sera from mice immunized with the BV HER1+HER2 were diluted 1:200 and incubated for 20 min with 10.sup.5 cells from tumor lines that co-express the HER1 and HER2 receptors. Lines that do not have increased expression of any of these receptors were used: H125, derived from lung carcinoma; H292, derived from lung carcinoma; PC3, derived from prostate carcinoma. In addition, lines with increased expression of HER1 were used: A431, derived from vulvar epithelial carcinoma; MDAMB468, derived from breast carcinoma and lines with increased expression of HER2: SKBR3, derived from breast carcinoma; MCF7/HER2 derived from breast carcinoma transfected with HER2; SKOV3, derived from ovarian carcinoma. The KRAS mutated lung carcinoma A549 cell line was also used. The Raji line, derived from Burkitt's lymphoma, was used as a negative control of receptor expression. The pre-immune sera were used as negative specificity controls while nimotuzumab MAb, specific for HER1, and trastuzumab MAb, specific for HER2, were used as positive controls. The recognition was evaluated by flow cytometry. FIG. 1 shows that the sera generated by the BV recognized all the cell lines evaluated, and the highest intensity of the recognition (corresponding to the highest mean fluorescence intensity (MFI)), was observed in the lines with increased expression of some of the receptors.

Example 2. The PAbs of the Immune Serum Induced by the HER1+HER2 BV Inhibit the Viability of Tumor Lines with Differential Expression of HER1 and HER2

[0051] The Effect is More Marked in Lines that do not Show Increased Expression of these Receptors.

[0052] To evaluate the effect of immune sera on lines with different expression levels of HER1 and HER2 as shown in Table 1, these levels were first corroborated using the mAbs trastuzumab and nimotuzumab, specific for HER2 and HER1 respectively. For this, the cells of the human tumor lines: A431, H125, H292, SKOV3 and SKBR3 were incubated for 20 min with the above-mentioned mAbs and the MFI was measured. The overlapping histograms of the recognition of the MAbs specific for HER1 and HER2 are shown in FIG. 2A. It has been reported in the literature that the flow cytometry of staining cell lines and the IHC of the patient's tumors are associated (Helfrich B A, et al., (2006) Clin Cancer Res; 12 (23) December 1; 7117-25), which allows us to extrapolate our results.

[0053] Immune sera from mice immunized with HER1+HER2 BV, diluted 1:20 and heated for 30 min at 56 C. to inactivate the proteins of the complement, were incubated with the tumor lines previously evaluated. The incubation with the sera was carried out for 96 h and the cell viability was determined by the MTT colorimetric method. As control of maximum viability (100%), cells without treatment of each of these lines and incubated under culture conditions were used. The cells treated with a mixture of pre-immune sera (1:20), were considered as negative control of induction of cytotoxicity, associated with the inhibition of HER1 and HER2 while AG1478 tyrosine kinase inhibitor, at a concentration of 10 M was used as positive control. The immune sera showed impact on the viability of all tumor lines as compared with those treated with the pre-immune sera in which no effect was observed (FIG. 2B). However, the most marked effect on cell viability was obtained on lines that do not show increased expression of any of the HER1 and HER2 receptors: lines H292 and H125, FIG. 2C.

TABLE-US-00001 TABLE 1 Levels of expression of HER1 and HER2 in the tumor lines evaluated. Expression Expression Cell lines levels of HER1 IMF levels of HER2 IMF A431 +++ 153.27 + 1.67 H125 ++ 61.77 + 1.98 H292 ++ 17.92 + 2.3 SKOV3 + 5.7 +++ 31.8 SKBR3 + 5.03 +++ 50.32

Example 3. The PAbs of the Immune Serum Induced by HER1+HER2 BV Inhibit the Activation of HER1 and HER2 Receptors and Proteins of Signaling Cascades

[0054] Sera from mice immunized with the BV was diluted 1:100 and incubated with cells of the H292 tumor line that co-express the HER1 and HER2 receptors but does not have increased expression of any of these receptors, for 30 min, 8 h and 24 h. The cells were stimulated with 100 ng/mL of EGF for 10 min to induce the activation of HER1 and of the HER2 receptors that were forming heterodimers with HER1. Subsequently, the treated cells were lysed. The effect of the immune sera on the inhibition of the phosphorylation of HER1, HER2, and proteins of the signaling cascades Erk1/Erk2 (MAPK), Akt and STAT3, was determined by means of a Western Blot assay, by the use of antibodies specific for the detection of said phosphorylated proteins. In this assay, the untreated cells were used as negative control of phosphorylation and the cells treated with EGF as positive control. The AG1478 tyrosine kinase inhibitor at 10 M was used as positive inhibition control and the pre-immune serum was used as a negative control of the specificity of the immune serum. In FIG. 3 it is observed that the sera containing the PAbs generated by the BV inhibited the activation of the HER1 and HER2 receptors, measured in terms of phosphorylation, from as early as 30 min, inhibition that increased markedly with the increase in the treatment time up to 8 h and 24 h. In addition, PAbs markedly inhibited the phosphorylation of Akt and STAT3 proteins, which are key proteins in signal transduction since they are inhibitors of apoptosis and inducers of inflammation, at 8 hours after treatment. Likewise, after 24 h, the total inhibition of the Erk1/Erk2 proteins of the MAPK pathway, inducers of cell proliferation, was observed.

Example 4. The PAbs of the Immune Serum Induced by the HER1+HER2 BV Cause Degradation of the HER1 and HER2 Receptors

[0055] Cells of the H292 tumor line were incubated with mixtures of immune sera (diluted 1:100) induced by HER1+HER2 BV for 30 min, 1, 8 or 24 h. The pre-immune serum was used as negative control in this assay. The expression levels of both receptors at the different times were determined by Western Blot. In FIG. 4 it is observed that the total expression levels of the HER1 and HER2 receptors decreased with the increase of the incubation time with the immune sera, as compared to the cells treated with the pre-immune sera. At 24 hours a total degradation of both receptors was observed.

Example 5. Comparison of the Capacity of the PAbs of the Immune Sera with that of the mAbs Nimotuzumab, Trastuzumab and their Combinations of Inhibiting the Viability of Tumor Lines with Low Expression of the HER1 and HER2 Receptors

[0056] Cells of the H292 tumor line were incubated for 96 h with mixtures of immune sera (diluted 1:100) induced by HER1+HER2 BV. Cell viability was determined by the MTT method. The pre-immune serum was uses as a negative control in this trial. Mitomycin C was used as a positive control of cytotoxicity. The effect of the immune sera was compared with that induced by nimotuzumab mAb, trastuzumab MAb and the mixture of both antibodies. FIG. 5 shows that the immune sera of the HER1+HER2 BV had a greater effect on the viability of the tumor cells than the mAbs and their combination.

Example 6. Comparison of the Capacity of the PAbs of the Immune Sera with that of Nimotuzumab, Cetuximab and Trastuzumab mAbs to Inhibit the Activation of the HER1 and HER2 Receptors

[0057] Sera from mice immunized with HER1+HER2 BV were diluted 1:100 and incubated for 1 h with cells from the tumor lines H125 and H292 that co-express the HER1 and HER2 receptors but have no increased expression of any of these receptors. This treatment was compared with the treatment with nimotuzumab and cetuximab MAbs, specific for HER1 and with trastuzumab MAb, specific for HER2. The cells were stimulated with 100 ng/mL of EGF for 10 min to induce the activation of HER1 and of the HER2 receptors that were forming heterodimers with HER1. Subsequently, the treated cells were lysed. Inhibition of receptor activation was measured by Western Blot. Cells treated with EGF were used as positive phosphorylation control. The AG1478 tyrosine kinase inhibitor was used as positive inhibition control and the pre-immune serum was used as a negative control of the specificity of the immune serum. FIG. 6 shows that the immune sera of the BV after 1 h of treatment had inhibited more the activation of HER1, measured in terms of phosphorylation, than the MAbs nimotuzumab and cetuximab. Likewise, they inhibited more the activation of HER2 than the MAb trastuzumab.

Example 7. Comparison of the Capacity of the PAbs Generated by the HER1+HER2 BV Against that of the AcMs Nimotuzumab, Cetuximab and Trastuzumab to Degrade the HER1 and HER2 Receptors

[0058] PAbs from the sera of mice immunized with HER1+HER2 BV, at a concentration of 10 g/mL, were incubated with cells from the H292 tumor line that co-express the HER1 and HER2 receptors but have no increased expression of any of these receptors. This treatment was compared with the treatment with nimotuzumab and cetuximab MAbs, specific for HER1, at the concentration of 10 g/mL and with cells incubation time of 24 h, and with trastuzumab MAb, specific for HER2, at a concentration of 1 g/mL and with cells incubation time of 1 h. It was also compared against the combination treatment with nimotuzumab and trastuzumab MAbs, and the combination treatment with the cetuximab and trastuzumab MAbs. In both combinations, the concentration of nimtozumab and cetuximab was 10 ng/mL and that of trastuzumab was 1 g/mL. The pre-immune serum was used as negative control of specificity. The cells were stimulated with 100 ng/mL of EGF for 10 minutes to induce the activation of HER1 and of the HER2 receptors that were forming heterodimers with HER1. Subsequently, the treated cells were lysed. The degraded receptors were detected by means of the ELISA technique, using a kit of reagents, Quantikine ELISA Human EGFR/ErbB1 Immunoassay to determine the degradation of HER1 and the DuoSet ELISA DEVELOPMENT SYSTEM Human ErbB2/Her2 to determine the degradation of HER2. FIG. 7A shows that the PAbs of the HER1+HER2 BV after 24 h of treatment had degraded more HER1 than nimotuzumab, cetuximab, and tratuzumab MAbs alone and combined. In FIG. 7B it is observed that the PAbs of the HER1+HER2 BV, after 1 h of treatment, degraded more HER2 than nimotuzumab, cetuximab, and tratuzumab MAbs and their combinations.

Example 8. Comparison of the Effect Induced by the PAbs of the HER1+HER2 BV on the Viability of the KRAS Mutated Lung Carcinoma A549 Cell Line, Against the Effect of Nimotuzumab, Cetuximab, Trastuzumab mAbs, and their Combinations

[0059] The PAbs from the serum of mice immunized with the HER1+HER2 BV (10 g/mL), were incubated for 72 h, with cells from the KRAS mutated lung carcinoma A549 cell line. Cell viability was determined by the MTT method. This treatment was compared against that with nimotuzumab and cetuximab MAbs, at a concentration of 10 g/mL, and against that with MAb trastuzumab, at a concentration of 1 g/mL. It was also compared with the combination treatment of nimotuzumab and trastuzumab MAbs, and the combination treatment of cetuximab and trastuzumab MAbs. In both combinations, the concentration of nimotuzumab and cetuximab was 10 g/mL and that of trastuzumab was 1 g/mL. As a negative control of the specificity of the PAbs generated by the HER1+HER2 BV, antibodies from the pre-immune sera were used. The % of viable cells resulting from the treatment with the PAbs was normalized against the negative control. FIG. 8 shows that the BV PAbs after 72 h of treatment inhibited the viability of the tumor cells by 40%, whereas cetuximab, nimotuzumab and trastuzumab inhibited only between 20% and 10%. The combinations had no effect on the viability of A549 cells.

Example 9. Comparison of the Effect Induced by the PAbs of the HER1+HER2 BV on the Degradation of HER1 and HER2, Against the Effect Induced by the Nimotuzumab, Cetuximab, Trastuzumab MAbs, and their Combination

[0060] PAbs from the sera of mice immunized with HER1+HER2 BV, at a concentration of 10 g/mL, were incubated for 24 h, with cells from the KRAS mutated lung carcinoma A549 cell line. This treatment was compared with that with nimotuzumab and cetuximab MAbs, at the 10 g/mL concentration, and against that with trastuzumab MAb, at a concentration of 1 g/mL. It was also compared with the combination treatment with nimotuzumab and trastuzumab MAbs, and the combination treatment with cetuximab and trastuzumab MAbs. In both combinations, the concentration of nimtozumab and cetuximab was 10 g/mL and that of trastuzumab was 1 g/mL. As a negative control of specificity were used the pre-immune sera. The cells were stimulated with 100 ng/mL of EGF for 10 minutes to induce the activation of HER1 and of the HER2 receptors that were forming heterodimers with HER1. Subsequently, the treated cells were lysed. The degraded HER1 receptors were detected by the ELISA technique, Quantikine ELISA Human EGFR/ErbB1 Immunoassay. In FIG. 9 it is observed that the PAbs induced by HER1+HER2 BV after 24 h of treatment had degraded more HER1 than nimotuzumab and cetuximab MAbs, alone or combined with trastuzumab MAb.