Pharmaceutical compositions for the treatment of tumors expressing EGFR and GM3 N-glycolyl ganglioside (NeuGcGM3)

Abstract

The present invention relates to pharmaceutical compositions for the treatment of malignant tumors. Particularly those tumors that express EGFR and GM3 N-glycolyl ganglioside targets to enhance the therapeutic effect produced by separated therapies against these targets. The pharmaceutical compositions of the invention include antibodies and/or vaccines against each of the targets. Additionally the present invention relates to methods for applying the compositions of the invention.

Claims

1. A method for retarding tumor growth of a tumor that co-expresses EGFR and N-glycolyl GM3 (NeuGcGM3) ganglioside targets in a patient in need thereof, comprising: (a) administering to a patient a medicament comprising a monoclonal antibody (MAb) directed against EGFR and, (b) administering to the same patient a medicament comprising a vaccine against NeuGcGM3 ganglioside.

2. The method according to claim 1, wherein (a) said MAb directed against EGFR is administered to said patient first and said vaccine against said NeuGcGM3 ganglioside is subsequently administered, or (b) said vaccine against said NeuGcGM3 ganglioside is administered to said patient first and said MAb against EGFR is subsequently administered.

3. The method according to claim 1, wherein said MAb directed against EGFR and said vaccine against said NeuGcGM3 ganglioside are administered simultaneously, staggered or alternately to said patient.

4. The method according to claim 1, wherein the vaccine against NeuGcGM3 ganglioside is NeuGcGM3/VSSP vaccine or anti-idiotypic Racotumumab/Alumina vaccine.

5. The method according to claim 1, wherein the dosage of the Mab is in the range from approximately 100 to 400 mg.

6. The method according to claim 4, wherein the dose of the NeuGcGM3 vaccine or the anti-idiotypic vaccine is in the range from approximately 0.1 to 2 mg.

7. The method according to claim 1, wherein said MAb is Nimotuzumab Mab.

8. The method according to claim 3, wherein said MAb and said vaccine against said NeuGcGM3 ganglioside are administered simultaneously in the form of a composition.

9. The method according to claim 1, wherein the vaccine has NeuGcGM3/VSSP as active principle.

10. The method according to claim 1, wherein the vaccine is Racotumumab anti-idiotypic vaccine adjuvated with alumina.

11. The method according to claim 1, wherein said patient is suffering from a tumor that co-expresses EGFR and NeuGcGM3 ganglioside targets.

12. The method according to claim 11, wherein said patient is suffering from a cancer selected from the group consisting of lung, breast, digestive system, urogenital system and sarcoma tumors derived from neuroectodermic tissue, and lymphoproliferative disorders.

13. The method according to claim 1, further comprising treating said patient with conventional radiotherapy.

14. The method according to claim 1, further comprising treating said patient with conventional chemotherapy.

15. The method according to claim 1, further comprising treating said patient with conventional radiotherapy and chemotherapy.

16. The method according to claim 1, wherein the patient experiences at least a 37% increase in survival percentage after 24 months of administration of the Mab directed against EGFR and the vaccine against NeuGcGM3.

17. The method according to claim 1, wherein there is less than 10% fall-off in survival of human cancer patients after 24 months of administration of the MAb against EGFR and the vaccine against NeuGcGM3 ganglioside compared to 12 months of administration of the MAb against EGFR and the vaccine against NeuGcGM3 ganglioside.

18. The method of claim 17, wherein the less than 10% fall-off in survival occurs in lung cancer patients.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. The EGFR is co-localized with NeuGcGM3 ganglioside in different murine tumors. Co-localization (C and F) of EGFR and NeuGcGM3 ganglioside in the lung metastases induced in Lewis epidermoid carcinoma (A-C) and murine myeloma P3-X63-Ag.8653 (D-F).

(2) FIG. 2. Anti-EGFR therapy (7A7 MAb) in combination with NeuGcGM3 (NeuGcGM3/VSSP) anti-ganglioside vaccine synergistically increases the survival of C57BL/6 mice bearing Lewis lung carcinoma.

(3) FIG. 3. Objective response to passive therapy against EGFR (Nimotuzumab) and NeuGcGM3 (Racotumumab/Alumina) anti-ganglioside vaccine in a patient with a non-small cell lung carcinoma.

(4) FIG. 4. Surprising clinical response to anti-EGFR (Nimotuzumab) in combination with NeuGcGM3 (NeuGcGM3/VSSP) ganglioside vaccine therapies in a patient with retroperitoneal-peripancreatic hemangiopericytoma.

EXAMPLES

Example 1: Measuring EGFR and NeuGcGM3 Ganglioside Coexpression in Different Human Tumors

(5) The tumor samples were fixed in neutral buffered formalin and processed by paraffin inclusion technique, which is known to those skilled in the art.

(6) The tissue sections obtained which had a thickness of 5 microns were maintained at 60° C. for 30 min, deparaffinized and rehydrated in a series of decreasing alcohols, kept in distilled water for 10 minutes and washed with TBS for 5 minutes. The reactivity of total tissue protein was blocked with a commercially available solution (Dako, Carpinteria, Calif., USA) for 30 min. The EGFR was immunodetected using the ior egf/R3m (20 μg/ml) for 1 hr. The immunorecognition of NeuGcGM3 was performed using 14F7 (MAb 20 μg/ml) for 30 min. After reaction of the primary antibodies, in both cases, the detection system used was Dako, LSABR Peroxidase System, DAB (Dako, Carpinteria, Calif., USA). Tissue sections were dehydrated and Mayers hematoxylin contrast was used (Dako, Carpinteria, Calif., USA). The negative control was obtained by substituting the primary antibody (Ior egf/R3m MAb or 14F7 MAb) by TBS wash solution (1×) and breast ductal carcinoma was used as a positive control.

(7) The immunorecognition of the EGFR was located on the cytoplasmic membrane of tumor cells and the NeuGcGM3 ganglioside was detected intracytoplasmic and/or in the cytoplasmic membrane of cells.

(8) Double staining: The EGFR was detected with Ior egf/R3m MAb (anti-EGFR) and then incubated with FITC-conjugated streptavidin (Dako, Carpinteria, Calif., USA). NeuGcGM3 was detected with biotinylated 14F7 MAb and subsequently with anti-murine IgG antibody conjugated to rhodamine (Dako, Carpinteria, Calif., USA). Co-expression of both molecules in tumor cells were identified in yellow in the images. Tissue sections were digitized and analyzed using a camera attached to an Olympus BX51 fluorescence microscope (Olympus, Japan). For the analysis of the digitized images ImageJ image processor version 1.43u was used.

(9) Table 1 shows tumors from different localizations wherein EGFR and NeuGcGM3 ganglioside are co-expressed.

(10) Intensity: −negative +weak ++moderate, +++intense.

(11) TABLE-US-00001 EGFR NeuGcGM3 Double positive Histological Positive Intensity Positive Intensity Positive Intensity types cases (%) Range cases (%) Range cases (%) Range Respiratory system NSCLC 8/10 (80)  ++/+++ 6/10 (60)   +/+++ 6/10 (60)  +/+++ Digestive system Stomach (ADC) 3/3 (100) ++/+++ .sup. 2/3 (66.6) ++/+++ 2/3 66.6) +/+++ Colorectal (ADC) 4/4 (100)  +/+++ 4/4 100)  +/+++  4/4 (100) ++ Pancreas (ADC) 3/4 (75)  ++/+++ 2/4 (50) ++/+++ 2/4 (50) ++ Liver 4/4 (100) ++/+++ 3/4 (75) ++/+++ 3/4 (75) +/+++ Urogenital system Bladder 2/2 (100) ++/+++ 1/2 (50) ++ 1/2 (50) ++ Nervous system Glioblastoma 5/5 (100) +++ 3/5 (60) ++ 3/5 (60) ++ multiforme Sarcomas  2/3 (66.6) +/++ .sup. 1/3 (33.3) + .sup. 1/3 (33.3) + Haemopoietic system Non-Hodgkin  2/3 (66.6) + .sup. 2/3 (66.6) + .sup. 2/3 (66.6) + lymphoma

Example 2: Coexpression and Co-Localization Measurement of EGFR and NeuGcGM3 Ganglioside in Murine Tumor Models

(12) Murine tumor models used were Lewis lung carcinoma (3LL-D122), and Myeloma P3-X63-Ag8.653 (X63).

(13) Double staining was performed to determine the co-expression/co-localization. The immunorecognition of EGFR was performed by incubation with 7A7 MAb (20 μg/ml), biotinylated, for 1 hr, followed by incubation with FITC-conjugated Streptavidin (Dako, Carpinteria, Calif., USA). Immunostaining of NeuGcGM3 was determined by incubation with 14F7 MAb (20 μg/ml), for 30 min, followed by incubation with an anti-murine IgG antibody conjugated to rhodamine (Dako, Carpinteria, Calif., USA). The negative control was obtained by replacing the primary antibody (7A7 MAb or 14F7 MAb) by TBS wash solution (1×). The co-expression was determined by using an Olympus BX51 fluorescence microscope (Olympus, Japan) and analysis of the digitized images using ImageJ image processor version 1.43u. The co-localization was determined by confocal laser microscope Flouview FV500 (Olympus, Japan). FIG. 1 shows the co-localization of EGFR and NeuGcGM3 in tumor samples from various murine models. The EGFR was identified with biotinylated 7A7 MAb followed by FITC-coupled streptavidin. The yellow color identifies the co-localization (C and F) of both molecules in tumor cells of murine models by color overlay.

Example 3: Measurement of Survival of C57BL/6 Mice Bearing Lewis Lung Carcinoma Treated with NeuGcGM3/VSSP Vaccine and 7A7 mAb

(14) Spontaneous Metastasis Model.

(15) The animals were inoculated in the right footpad with 2×10.sup.5 cells of Lewis epidermoid carcinoma (3LL-D122), in a volume of 0.05 ml, which corresponded to day 0 of the experimental protocol. On day 3 of the experiment the animals were randomized into four experimental groups of 10 animals each. On day 24, when the tumor reached a volume of 8-9 mm primary tumor surgery was performed. From day 48, observation of the clinical status of the animals began. Survival data were analyzed using the Log-rank test, p<0.05, and displayed on a Kaplan-Meier plot. The result includes three experiments performed on equal conditions.

(16) Experimental Groups

(17) Untreated Control. (T)

(18) Passive anti-EGFR therapy: 7A7 MAb, intravenously at a dose of 56 μg in 200 μl of Saline Solution, on days 3, 5, 7, 9, 31, 33 and 35.

(19) Active anti-NeuGcGM3 therapy: NeuGcGM3/VSSP vaccine, subcutaneously at a dose of 200 μg in 200 μl, on days 7, 21, 35 and 47.

(20) Simultaneous administration of anti-EGFR and anti-NeuGcGM3 therapies as described for groups 2 and 3.

(21) FIG. 2a shows that the simultaneous administration of anti-EGFR and anti-NeuGcGM3 (group 4) therapies on the lung metastasis model induced with Lewis lung carcinoma, increased survival of the animals by 60% compared with the other experimental groups. Surviving animals (one in group 4) were sacrificed one week after the end of the experiment and their lungs were removed. Macroscopic analysis showed that only one animal showed two lung metastases, noticing also that the rest the lungs of the animals were normal. (FIG. 2b). This result demonstrates a strong synergy of antitumor activity of anti-EGFR and anti-NeuGcGM3 therapies studied in this murine tumor model wherein EGFR and NeuGcGM3 are co-localizated (see Example 2)

Example 4: Measurement of Survival of Cancer Patients Undergoing Therapy with Nimotuzumab Antibody and Racotumumab/Alumina Vaccine

(22) Given the finding on the frequent co-localization/co-expression of therapeutic targets EGFR and NeuGcGM3 ganglioside in samples of human lung tumor (Example 1); and the preclinical evidence shown in Example 3, we proceeded to study simultaneous treatment with anti-EGFR (Nimotuzumab antibody) and anti-ganglioside (Racotumumab/Alumina anti-idiotypic vaccine) therapies in lung cancer patients who had received the standard therapy for each tumor site and who had already disease progression.

(23) Table 2 shows the test results from expanded use program (compassionate) of Racotumomab (1E10/Alumina) vaccine as single therapy or in combination with Nimotuzumab. Survival of cancer patients with non-small cell lung cancer (NSCLC) in advanced stages (Recurrent and/or Metastatic) is observed on it. It should be noted that patients on this study had received all established standard treatment lines and were only candidates for palliative, non onco-specific, therapy and that at the time of inclusion in the trial they had disease progression. In the group of patients that received therapy simultaneously against both targets a significant increase in overall survival at two years of treatment was observed, as compared with those who received only the monotherapies.

(24) TABLE-US-00002 TABLE 2 Increased survival of patients with non-small cell lung cancer treated with anti-EGFR (Nimotuzumab antibody) and anti-ganglioside (Racotumumab/Alumina anti-idiotypic vaccine) therapies at two years of treatment. Treatment Groups SV at 12 m (%) SV at 24 m (%) Racotumumab/Alumina 42.7 16.1 (n.sub.=86) Nimotuzumab (n.sub.=165) 36 21.4 Racotumumab/Alumina + 40.7 37.7 Nimotuzumab (n.sub.=88)

(25) FIG. 3 shows the objective response to passive therapy against EGFR (Nimotuzumab) and NeuGcGM3 anti-ganglioside vaccine (Racotumomab/Alumina) in a patient with NSCLC that, as mentioned above, had received all established standard treatments lines, which was candidate only for palliative, non onco-specific, therapy and that at the time of inclusion in the trial had disease progression. FIG. 3A shows the localization and extent of the tumor at the time of diagnosis. FIG. 3B shows the results from Computed Tomography (CT) at two years after treatment. In the latter figure only areas of fibroblastic response with areas of increased transparency in relation with bullae of emphysema that can be seen and no lung tumor lesion is observed. The same radiographic image remains three years later. This result indicates a surprising complete remission of a very advanced tumor as a result of therapy.

Example 5: Measurement of Clinical Response in a Patient with Retroperitoneal-Peripancreatic Hemangiopericytoma Treated with the Nimotuzumab Antibody and NeuGcGM3/VSSP Vaccine

(26) FIG. 4 shows a sequential computed tomography of the abdomen of a patient with retroperitoneal-peripancreatic hemangiopericytoma (soft tissue tumor), with no response to radiotherapy and chemotherapy. The patient received anti-EGFR (Nimotuzumab) and anti-ganglioside (NeuGcGM3/VSSP vaccine) therapies 18 months after the initial diagnosis. The patient had severe pain and a throbbing tumor mass in the periumbilical region and weight loss of more than 15 kilos. FIGS. 4A and B correspond to the start time of therapies; C and D correspond to the evaluation after three years. Three years after application of treatment a stabilization of the disease can be observed, as evidenced by both images wherein the tumor size remains the same. Moreover, in the 72-month evaluation, patient maintains an excellent quality of life and is able to continue his working life. A tendency towards reduction of the tumor mass can also be seen. In summary, there is a surprising clinical benefit as response to the treatment.

(27) Cancer patients treated with anti-EGFR and anti-ganglioside immunotherapy, as described in this invention, exhibit excellent tolerance (no significant toxicity) for long periods of treatment. The therapy promotes stabilization of the disease, increasing the quality of life, time to progression and overall survival of patients. The therapy described above has a superior effect to that observed in patients receiving traditional anti-EGFR or anti-ganglioside monotherapies.