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Title:
ELEVATED BIOMARKER EXPRESSION IN LUNG CANCER PATIENTS RESPONDING TO TREATMENT WITH MUC-1 LIPOPEPTIDE VACCINES
Document Type and Number:
WIPO Patent Application WO/2015/058831
Kind Code:
A1
Abstract:
The invention is directed to the treatment of lung cancer, preferably non-small cell lung cancer (NSCLC) by means of a combination therapy comprising chemo- radiotherapy and vaccination with muc-1 lipopeptides, preferably tecemotide (L-BLP- 25). The invention relates, above all, to specific biomarkers which are overexpressed and released into the peripheral blood of patients suffering from lung cancer, preferably NSLC, who respond to the vaccination treatment with said drug in combination with chemo-radiotherapy, preferably concurrent chemo-radiotherapy prior to vaccination, compared to respective patients who do not respond or do weakly respond to said treatment.

Inventors:
SCHROEDER, Andreas (Haeuselgasse 15, Heidelberg, 69124, DE)
HELWIG, Christoph (Gerbergasse 11A, Seligenstadt, 63500, DE)
Application Number:
EP2014/002677
Publication Date:
April 30, 2015
Filing Date:
October 01, 2014
Export Citation:
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Assignee:
MERCK PATENT GMBH (Frankfurter Strasse 250, Darmstadt, 64293, DE)
International Classes:
G01N33/574; G01N33/68
Domestic Patent References:
WO2005112546A22005-12-01
WO1998050527A11998-11-12
Other References:
MITCHELL ET AL: "Tecemotide (L-BLP25) in unresectable stage III non-small cell lung cancer in the phase III START study: Further endpoint and exploratory biomarker results", IASLC, 15TH WORLD CONFERENCE ON LUNG CANCER, OCTOBER 27 - OCTOBER 30, 2013, O02.02, 10 October 2013 (2013-10-10), Sydney, Australia, XP002734450, Retrieved from the Internet [retrieved on 20150114]
GREGORY T WURZ ET AL: "Antitumor effects of L-BLP25 Antigen-Specific tumor immunotherapy in a novel human MUC1 transgenic lung cancer mouse model", JOURNAL OF TRANSLATIONAL MEDICINE, BIOMED CENTRAL, LONDON, GB, vol. 11, no. 1, 13 March 2013 (2013-03-13), pages 64, XP021142105, ISSN: 1479-5876, DOI: 10.1186/1479-5876-11-64
SHEPERD ET AL: "Updated analysis and secondary endpoints with tecemotide (L-BLP25) in unresectable stage III non-small cell lung cancer in the phase III START study", EUROPEAN CANCER CONGRESS, 27 SEPTEMBER - 1 OCTOBER 2013, 3419, 27 September 2013 (2013-09-27), pages 1 - 1, XP002734451, Retrieved from the Internet [retrieved on 20150113]
GRAUS F ET AL: "ANTI-HU ANTIBODIES IN PATIENTS WITH SMALL-CELL LUNG CANCER: ASSOCIATION WITH COMPLETE RESPONSE TO THERAPY AND IMPROVED SURVIVAL", JOURNAL OF CLINICAL ONCOLOGY, AMERICAN SOCIETY OF CLINICAL ONCOLOGY, US, vol. 15, no. 8, 1 August 1997 (1997-08-01), pages 2866 - 2872, XP000995600, ISSN: 0732-183X
MENGYING YAO ET AL: "Overexpression of MUC1 Enhances Proangiogenic Activity of Non-Small-Cell Lung Cancer Cells Through Activation of Akt and Extracellular Signal-regulated Kinase Pathways", LUNG, SPRINGER-VERLAG, US, vol. 189, no. 6, 1 October 2011 (2011-10-01), pages 453 - 460, XP019981096, ISSN: 1432-1750, DOI: 10.1007/S00408-011-9327-Y
BEARZ A ET AL: "MUC-1 (CA 15-3 antigen) as a highly reliable predictor of response to EGFR inhibitors in patients with bronchioloalveolar carcinoma: an experience on 26 patients", INTERNATIONAL JOURNAL OF BIOLOGICAL MARKERS, vol. 22, no. 4, October 2007 (2007-10-01), pages 307 - 311, XP002734452, ISSN: 0393-6155
BARND, PROC NATL ACAD SCI USA., vol. 86, 1989, pages 7159 - 7163
CHOI ET AL., BLOOD, vol. 105, 2005, pages 2132 - 2134
GAO ET AL., INT J ONCOL., vol. 35, 2009, pages 337 - 345
RAINA ET AL., MOL CANCER THER., vol. 10, 2011, pages 806 - 816
MEHTA ET AL., CIN CANCER RES., vol. 18, 2012, pages 2861 - 2871
BUTTS ET AL., J CANCER RES CLIN ONCOL., vol. 137, 2011, pages 1337 - 1342
FERNANDEZ-MADRID ET AL.: "Antinuclear Antibodies as Potential Markers of Lung Cancer", CLINICAL CANCER RESEARCH 1393, vol. 5, June 1999 (1999-06-01), pages 1393 - 1400, XP055161685, DOI: doi:10.1016/B978-044450331-2/50017-5
ISHIKAWA ET AL., INT. J. CANCER, vol. 122, 2008, pages 2612 - 2620
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Claims:
PATENT CLAIMS:

1. A liposomal formulation comprising a lipopeptide based on the muc-1 core

repeating unit selected from the group consisting of the amino acid sequences:

STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID No. I) or

STAPPAHGVTSAPDTRPAPGSTAPP-K-palmitoyl- (G) (SEQ ID No. II)

for use in a method of treatment of lung cancer in combination with chemo- radiotherapy, wherein the treatment is effective and clinically responsive to a patient suffering from said lung cancer, when in a sample of peripheral blood of the patient before starting treatment

(i) the titer of anti-nuclear antibodies (ANA) is greater than 1 :40, or

(ii) the concentration of soluble MUC-1 (sMUC1) is > 22 lU/mL

2. A liposomal formulation for use according to claim 1 , wherein

(i) the titer of anti-nuclear antibodies (ANA) is greater than 1:40, and

(ii) the concentration of soluble MUC-1 (sMUC1) is > 22 lU/mL.

3. A liposomal formulation for use according to claim 1 or 2, wherein the ANA titer is > 1: 160, and the sMUC1 concentration is > 32 lU/mL.

4. A liposomal formulation for use according to any of the claims 1 - 3, wherein the treatment causes, when applied to a cohort of responding patients, an overall- survival (OS), which is statistically prolonged between 15 - 50%, and an adjusted hazard ratio < 0.60.

5. A liposomal formulation comprising a lipopeptide based on the muc-1 core

repeating unit selected from the group consisting of the amino acid sequences:

STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID o. I) ΟΓ

STAPPAHGVTSAPDTRPAPGSTAPP-K-palmitoyl - (G) (SEQ ID No. II)

for use in a method of treatment of lung cancer in combination with chemo- radiotherapy, wherein the treatment is contraindicated if the patient's peripheral blood elicits

(i) no antibodies (ANA), or an ANA titer of less than 1 : 20, or

(ii) a concentration of soluble MUC-1 (sMUC1) is < 20 lU/mL

6. A liposomal formulation for use according to claim 5, wherein

(i) the ANA titer is less than 1 : 20, and

(ii) a concentration of soluble MUC-1 (sMUC1) is < 20 lU/mL.

7. A liposomal formulation for use according to any of the claims 1 - 6, wherein the lung cancer is non-small lung cancer NSCLC.

8. A liposomal formulation for use according to claim 7, wherein the lung cancer is unresectable stage III NSCLC.

9. A liposomal formulation for use according to any of the claims 1 - 8, wherein treatment comprises concurrent chemo-radiotherapy followed by vaccination with the liposomal formulation.

10. A liposomal formulation for use according to any of the claims 1 - 9, wherein the chemotherapy comprises platinum-based chemotherapeutic agents.

11. A liposomal formulation for use according to any of the claims 1 - 10, further comprising an adjuvant. 12. The liposomal formulation for use according to claim 11 , wherein the adjuvant is selected from the group consisting of MPL(3-Odesacyl-4'-monophosphoryl lipid), Lipid A.which is part of the liposomal formulation.

13. The liposomal formulation for use according to claim 12, wherein the lipopetide is based on SEQ ID NO. 2 and the MPL-lipopetide liposomal formulation is designated as L-BLP25 (tecemotide).

14. An in-vitro method for predicting the likelihood that a patient will respond

therapeutically to a method of treating lung cancer by administering to a patient a liposomal formulation comprising a lipopeptide based on the muc-1 core repeating unit selected from the group consisting of the amino acid sequences: STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID o. I) or

STAPPAHGVTSAPDTRPAPGSTAPP-K-palmitoyl - (G) (SEQ ID No. II),

wherein the method comprises

(i) measuring from a peripheral blood sample of the diseased patient before starting the treatment with said lipopeptide, the amount or titer of anti-nuclear antibodies (ANA) and / or the amount of soluble MUC-1 (sMUCI) by means of common analytical tools and methods, and

(ii) selecting patients for which the administration with said drug is indicated and responsive when the titer of ANA is greater than 1 : 40, preferably 1 : 80, and/or the amount of sMUCI is greater than 22 lU/mL, preferably greater than 32 lU/ml; or

selecting patients for which the administration with said drug is contra-indicated and non-responsive when the titer of ANA is less than 1 : 40, preferably less than 1 : 20, and / or the amount of sMUCI is less than 22 lU/mL, preferably less than 20 lU/mL

15. An in-vitro method of claim 14, wherein the lung cancer is non-small lung cancer NSCLC.

16. An in-vitro method of claim 15, wherein the lung cancer is unresectable stage III NSCLC. 7. An in-vitro method according to any of the claims 14 - 16, wherein the treatment comprises concurrent chemo-radiotherapy followed by vaccination with the liposomal formulation.

18. An in-vitro method according to any of the claims 14 - 17, wherein the

chemotherapy comprises platinum-based chemotherapeutic agents. 19. An in-vitro method according to any of the claims 14 - 8, further comprising an adjuvant.

20. An in-vitro method according to any of the claims 14 - 19, wherein the adjuvant is selected from the group consisting of MPL(3-Odesacyl-4'-monophosphoryl lipid), Lipid A, which is part of the liposomal formulation. 21. An in-vitro method according to any of the claims 14 - 20, wherein the lipopetide is based on SEQ ID NO. 2 and the MPL-lipopetide liposomal formulation is designated as L-BLP25 (tecemotide).

22. Use of soluble MUC-1 (sMUCI) as selective diagnostic biomarker to predict ex- vivo the efficacy or benefit of the treatment with L-BLP25 (tecemotide) in patients suffering from stage III unresectable NSCLC who previously underwent chemoradiotherapy.

23. Use of sMUC1 according to claim 22, wherein samples of the peripheral blood of the patient are analyzed for sMUC1 by means of standard assays and

techniques.

24. Use of sMUC1 according to claim 23, wherein the treatment with L-BLP25 is

beneficial and therapeutically indicated for the patient, if the biomarker is present in the blood sample in a concentration greater than 25 lU/mL, preferably greater than 32 lU/mL 25. Use of sMUC1 according to claim 23, wherein the treatment with L-BLP25 is not effective and therapeutically contraindicated for the patient, if the biomarker is present in the blood sample in a concentration less than 20 lU/mL

26. Use of anti-nuclear antibodies (ANAs) as selective diagnostic biomarker to predict ex-vivo the efficacy of the treatment with L-BLP25 (tecemotide) in patients suffering from stage III unresectable NSCLC who previously underwent

chemoradiotherapy.

27. Use of ANAs according to claim 26, wherein samples of the peripheral blood of the patient are analyzed for ANAs by means of standard assays and techniques.

28. Use of ANAs according to claim 27, wherein the treatment with L-BLP25 is

beneficial and therapeutically indicated for the patient, if the biomarker is present in the blood sample with a titer greater than 1 : 80, preferably greater than 1 :160.

29. Use of ANAs according to claim 27, wherein the treatment with L-BLP25 is

beneficial and therapeutically indicated for the patient, if the biomarker is present in the blood sample with a titer less than 1 :40, preferably less than 1 :20.

Description:
ELEVATED BIOMARKER EXPRESSION IN LUNG CANCER PATIENTS RESPONDING TO TREATMENT WITH MUC-1 LIPOPEPTIDE VACCINES

FIELD OF THE INVENTION: The invention is directed to the treatment of lung cancer, preferably non-small cell lung cancer (NSCLC) by means of a combination therapy comprising chemo-radiotherapy and vaccination with muc-1 lipopeptides, preferably tecemotide (L-BLP-25). The invention relates, above all, to specific biomarkers which are overexpressed and released into the peripheral blood of patients suffering from lung cancer, preferably NSLC, who respond to the vaccination treatment with said drug in combination with chemo-radiotherapy, preferably concurrent chemo-radiotherapy prior to vaccination, compared to respective patients who do not respond or do weakly respond to said treatment.

BACKGROUND OF THE INVENTION

Lung cancer is the leading cause of cancer death in men, with an overall 5-year survival rate of approximately 10 to 15%. The limited efficacy and the toxicity associated with

chemotherapy for non-small cell lung cancer (NSCLC) has created a need for safer and more efficacious treatment options. With the identification of tumor-associated antibodies and antigens (TAA) in patients with lung cancer, immunotherapy has emerged as an attractive alternative. Mucin 1 (MUC1) is one such TAA that is an epithelial glycoprotein overexpressed in NSCLC. T-cells specific for antigenic epitopes of MUC1 that bind to HLA class I molecules, have been identified and isolated from the blood and bone marrow of cancer patients (Barnd et al., Proc Natl Acad Sci USA. 1989;86:7159-7163; Choi et al. Blood. 2005;105:2132-2134). MUC1 is a highly glycosylated transmembrane protein that is normally present on the luminal surface of secretory glands.1 In contrast to healthy glandular tissues, many adenocarcinomas (breast, ovary, pancreas, and lung) express a hypoglycosylated form of MUC1.

The immune-dominant peptides from the variable number of tandem repeat region of mucin (VNTR) are recognized by the cytotoxic T-lymphocytes (CTL), making MUC1 an attractive target for therapeutic intervention. The repeating peptide unit is built by 20 amino acid:

STAPPAHGVTSAPDTRPAPG.

A number of studies have shown that MUC1 may facilitate epithelial carcinogenesis. High MUC1 expression in tumors has been correlated with increased invasiveness, migration, and angiogenesis in ovarian and lung cancers. Depolarized expression of MUC1 has been related to poor prognosis in early stage NSCLC (Gao et al. Int J Oncol. 2009;35:337-345). Recent findings have indicated that NSCLC cells are dependent on the MUC1-C terminal cytoplasmic domain for both activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway and for survival (Raina et al. Mol Cancer Ther. 201 1 ; 10:806-816).

A number of studies are focused on devising techniques to effectively present UC1 as an immunogenic agent to stimulate a strong and highly specific immune response against target cells over-expressing MUC1. L-BLP25 is one such innovative liposomal antigen-specific cancer immunotherapy currently under development that contains 25 amino acids from the immunogenic tandem-repeat region of MUC1 (Mehta et al.,Clin Cancer Res. 2012; 18:2861- 2871): STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID No. 1).

L-BLP25 (tecemotide) is an active immunotherapeutic agent designed to induce a cellular immune response by targeting T-cell epitopes from the VNTR region of the MUC1 antigen associated with HLA class I molecules. Although NSCLC is historically regarded as a non- immunogenic cancer, L-BLP25 in phase II clinical trials has shown survival advantages with a remarkably low toxicity profile (WO 2005/112546; Butts et al.,J Cancer Res Clin Oncol.

2011 ;137:1337-1342). In these trials a single, low, intravenous dose (300mg/m 2 to a maximum of 600mg) of cyclophosphamide (CPA) is administered three days prior to immunotherapy. This procedure is thought to enhance delayed-type hypersensitivity humoral and cellular immune responses by reducing T-suppressor function. Although CPA lacks any significant activity in NSCLC, and the dose used in this setting is below that used in cytotoxic chemotherapy, it is currently believed that the observed antitumor effects following L-BLP25 therapy can be attributed to the immunomodulatory effects of CPA.

As described below in more detail it could be shown that in a comprehensive statistically based clinical trial, vaccination with tecemotide is effective in combination with chemo- radiotherapy if applied to a cohort of lung tumor patients, preferably, patients suffering from non-small cell lung cancer (NSCLC), and most preferably patients suffering from unresectable stage III NSCLC. Interestingly, the chemo-radiotherapy is much more effective if the chemo- radiotherapy approach is started before vaccination, and chemotherapy and radiotherapy are carried out concurrently/simultaneously or timely overlapping by at least 30 - 50% calculated of the chemotherapy duration. In contrast, and surprisingly, the efficacy of vaccination with said muc- lipopeptides as specified in this invention is strongly reduced and if any only slightly increased versus the same treatment with a placebo if the radiation therapy is started after completion of the chemotherapy or is timely overlapping with chemotherapy by less than 10% of the duration of the treatment with chemotherapeutic agents. The statistic overall survival time (OS) and the time of disease progression (TTP) of a patient group can be extended by up to 50%. The vaccination with said muc-1 lipopetide formulations of the invention after completion of a sequential chemo-radiotherapy provokes no significant effect with regard to OS TTP versus the administration of a placebo, whereas the same setting in a concurrent chemo-radiotherapy causes a prolongation of up to 60% compared to the respective placebo administration.

Although the efficacy of the treatment of lung cancer patients, especially NSCLC patients could be improved by administering to a patient via vaccination a MUC1 lipopeptide, preferably a liposomal formulation of tecemotide, by means of a specific treatment regimen comprising chemo-radiotherapy, wherein the preceding chemo-radiotherapy is carried out preferably concurrently, it had to be realized that some patients respond to the treatment much better than others. Therefore, there is a need to find out the reasons for that different patient's responsiveness to lung cancer, preferably NSCLC, against BLP25/tecemotide and related muc-1 lipopeptides, and to provide a clear technical guidance for the responsible physician before starting treatment with said drugs which patients or patient groups will elicit high efficacy to the treatment and for which patients or patient groups the proposed treatment is contra-indicated and should not be carried out for ethical and economic reasons.

SUMMARY OF THE INVENTION It was found by the inventors that patients suffering from lung cancer, especially from non- small cell lung cancer (NSCLC), preferably from unresectable stage III NSCLC are

responsive to the treatment of vaccination with drugs comprising the mic-1 core repeating unit, preferably by means of a lipopetide comprising liposomal formulation preferably in connection with an adjuvant, if the diseased cancer patient elicits elevated or increased levels of a soluble form of human MUC1 (sMUC1) and / or human antinuclear antibodies (ANA) in body fluids, preferably the peripheral blood of the untreated patient. These patients show statistically up to 60% more clinical /therapeutic responsiveness to the treatment as compared to diseased patients eliciting these molecules in low levels or low levels which can be related to healthy persons. Therefore, the invention is related to a liposomal formulation comprising a lipopeptide based on the muc-1 core repeating unit selected from the group consisting of the amino acid sequences: S APPAHGVTSAPDTRPAPGS APP (SEQ ID Νθ. I) ΟΓ

STAPPAHGVTSAPDTRPAPGSTAPP-K-palmitoyl- (G) (SEQ ID Νθ. II) for use in a method of treatment of lung cancer in combination with chemo-radiotherapy, wherein the treatment is effective and clinically responsive to a patient suffering from said lung cancer, when in a sample preferably from the peripheral blood of the untreated patient, which means before starting treatment, (i) the titer of anti-nuclear antibodies (ANA) is greater than 1 :20, 1 :40, 1 :80, 1 :160 and 1 :240, preferably greater than 1: 40, more preferably greater than 1 : 80, and most preferably greater than 1 :160 (note: the titer is the higher, the higher the second digit is)

or alternatively (ii) the concentration of soluble MUC-1 (sMUC1) is > 20 lU/mL, preferably > 22 lU/mL, more preferably > 25 lU/mL, and most preferably > 32

According to the invention, the ANA titers of responsive untreated (with respect to the MUC1- lipopeptide vaccine formulation as specified) patients (patients before starting the potential treatment with said drug) vary preferably between 1 : 20 and 1 : 240, preferably between :40 or 1:80 and 1 : 160 in the peripheral blood. Correspondingly, the ANA titers in the peripheral blood of patients which do not or not significantly respond therapeutically to the treatment with said drug, and for which the treatment is therefore contraindicated and not effective, elicit an ANA titer below 1 :20, preferably below 1 : 15 or even less than 1 :10.

According to the invention, the sMUC1 contents of responsive untreated (with respect to the MUC1-lipopeptide vaccine formulation as specified) patients (patients before starting the potential treatment with said drug) vary preferably between 20 lU/mL and 40 lU/mL, preferably between 22 lU/mL and 30 lU/ml in the peripheral blood. Correspondingly, the sMUC1 contents in the peripheral blood of patients which do not or not significantly respond therapeutically to the treatment with said drug, and for which the treatment is therefore contraindicated and not effective, elicit an amount of sMUC1 in the peripheral blood less than 20 lU/mL, preferably less than 5, and more preferably less than 10 lU/mL

The inventors found that therapeutically responsive patients having an enhanced ANA titer not necessarily also have an enhanced sMUC1 level in their blood. Only in a miner subpopulation of patients (less than 5%) the ANA titer as well as the sMUC1 concentration is increased.

A liposomal formulation comprising a lipopeptide based on the muc-1 core repeating unit selected from the group consisting of the amino acid sequences:

STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID No. I) or STAPPAHGVTSAPDTRPAPGSTAPP-K-palmitoyl- (G) (SEQ ID No. II) or specifically L- BP25 (tecemotide), which elicits clinical responsiveness in lung cancer patients

in the treatment of lung cancer in combination with chemo-radiotherapy causes, when applied to a cohort of responding patients, a statistic overall-survival (OS) of 15 - 50%, and an adjusted statistic hazard ratio < 0.60, preferably < 0.55, more preferably < 0.50.

In a specific embodiment of the invention the lung cancer is non-small lung cancer NSCLC, and preferably unresectable stage III NSCLC.

In another preferred embodiment of the invention the treatment comprises concurrent instead of sequential chemo-radiotherapy followed by vaccination with said liposomal formulation. The invention relates further to said use of said liposomal formulation, wherein the

chemotherapy is carried out by administering chemotherapeutic agents including at least one platinum based chemotherapeutic compound, preferably cisplatin or carboplatin. In addition further chemotherapeutic agents can be applied and may be helpful.

The invention is further related to said use of said liposomal formulation, wherein an adjuvant is applied together with the liposomal vaccine formulation. In a preferred embodiment, the adjuvant is part of the liposome that contains the muc-1 lipopetide or integrated into the liposome.

The liposomal formulation of the invention comprises preferably an adjuvant, which is selected from the group consisting of MPL(3-Odesacyl-4'-monophosphoryl lipid), Lipid A, or low-toxic variants of LPS. MPL is most preferred. The invention is specifically directed to a liposomal formulation, wherein the muc-1 lipopetide is based on SEQ ID NO. 2. The respective liposomal MPL-lipopetide formulation is designated as L-BLP25.

The liposomal formulation according to the invention is effective in vivo in patients suffering from lung cancer, preferably non-small cell lung cancer (NSCLC), and most preferably unresectable stage III NSCLC. Nonetheless, it cannot be excluded that the treatment as provided can be successfully used in the treatment of cancers different from lung cancer, such as breast or prostate cancer and the like.

According to the invention, the liposomal formulation can be administered in combination with at least a further pharmaceutically effective anti-cancer agent. Furthermore, the invention is related to a method of treating a patient suffering from lung cancer, preferably NSCLC, more preferably unresectable stage III NSCLC, comprising the following steps:

(i) applying chemo-radiotherapy to said patient, wherein said chemotherapy, preferably platinum-based chemotherapy, preferably cisplatin or carboplatin, and said radiotherapy is carried out concurrently or at least timely overlapping, preferably by at least 10% - 100%, preferably 20 - 100%, most preferably 70 - 100% related to the duration of the

chemotherapy, and

(ii) vaccinating said patient after completion of said chemo-radiotherapy every 5 th - 9 th day, preferably every 7 th day for at least 4 - 8 administrations, and every 35 th - 49 th day, preferably every 42 nd day for the following period, with a liposomal formulation comprising a lipopeptide based on the muc-1 core repeating unit selected from the group consisting of the amino acid sequences:

STAPPAHGV SAPDTRPAPGS APP (SEQ ID Νθ. I) ΟΓ

STAPPAHGVTSAPDTRPAPGSTAPP-K-palmitoyl- (G) (SEQ ID Νθ. II),

preferably together with an adjuvant and / or a further anti-cancer agent,

wherein said liposomal formulation is applied not later than 180 days, preferably not later than 140 days, and most preferably not later than 98 days after completion of said chemo- radiotherapy.

Furthermore, the invention is related to a method of extending the survival time of a patient suffering from non-small cell lung cancer (NSCLC), preferably unresectable stage III NSCLC treated with a liposomal formulation comprising a lipopeptide based on the muc-1 core repeating unit selected from the group consisting of the amino acid sequences:

STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID Νθ. I) ΟΓ STAPPAHGVTSAPDTRPAPGSTAPP-K- paimitoyi- (G) (SEQ ID No. II), by pre-treating the patient with concurrent or at least 10 - 95% timely overlapping chemo-radiotherapy which is completed at least 14 - 35 days, preferably 21 - 28 days before starting vaccination with said liposomal formulation but not later than 180 days, preferably not later than 140 days, preferably not later than 98 days, and most preferably not later than 84 - 98 days, wherein said extension is at least 15%, preferably at least 25%, compared to a respective treatment comprising an analogous sequential chemo-radiotherapy treatment, and at least 25%, preferably at least 35% compared to an analogous concurrent chemo-radiotherapy treatment, wherein a placebo is applied instead of the liposomal formulation, wherein radiotherapy is carried out by applying at least 40 Gy, preferably 50 - 120 Gy, more preferably 50 - 75 Gy of total radiation during chemo-radiotherapy, and chemotherapy is carried out by administering at least one platinum- based chemotherapeutic agent , selected from the group consisting of cisplatin and carboplatin together with an adjuvant, preferably MPL or Lipid A, and optionally an immune modulating agent, preferably cyclophosphamide, and /or a further anti-cancer agent, by at least two cycles, preferably 2 - 8 cycles, wherein one cycle is between 21 and 35 days, preferably between 21 and 28 days, and wherein the platinum-based chemotherapeutic agent is administered in daily, weekly or 2 - 5 weekly dose. Finally the invention is related to the use of L-BLP25 (tecetomide, Stimuvax (R) ) for the treatment of a patient suffering from unresectable stage III non-small cell lung cancer

(NSCLC) by means of a combination therapy including chemo-radiotherapy followed by vaccination of the patient with L-BLP25, wherein the initial chemo-radiotherapy is concurrent or at least 10 - 95%, preferably 50- 95% timely overlapping related to the duration of the chemotherapy, and the vaccination starts after completion of said chemo-radiotherapy not later than 98 days, preferably not later than 84 days, and wherein the chemotherapy is based on platinum-based chemotherapeutic agents, preferably cisplatin and carboplatin.

Conclusions:

While the primary endpoint of OS prolongation was not met, secondary endpoints PFS and TTF support a more favorable effect of tecemotide vs. placebo in patients treated with concurrent chemo/RT. Similar findings can be observed for OS, TTSP and TTP. Any further clinical investigation of tecemotide in locally advanced NSCLC should focus on patients who have received concurrent chemo/RT. ANA and sMUC1 warrant further investigation as potential predictive biomarkers. DETAILED DESCRIPTION OF THE INVENTION

The basic finding according to the invention is that two specific biomarkers could be identified preferably in the peripheral blood of lung cancer patients, preferably NSCLC patients, which are specific for the responsiveness of patients to the suggested treatment with a MUC1 lipopetide formulation, preferably L-BLP25 (tecetomide). These two biomarkers occurring in the blood of responsive patients by increased or significantly enhanced levels and

concentrations (whereas they are not or not significantly present in patients that do not respond) are soluble MUC-1 and anti-nuclear antibodies (ANAs).

A soluble form of MUC1 (sMUC1) was already detected in breast milk, peripheral blood, urine, and supernatants from cultures of MUC1 cancer cell lines and primary cancer cells. sMUC1 appears to represent a truncated form of MUC1 that lacks the cytoplasmic domain of membrane bound, full-length MUC. The mechanism by which sMUC1 is formed needs still to be clarified but may result from alternative splicing of the MUC1 gene product, leading to the production of a secreted form of MUC or from proteolytic cleavage of full-length, membrane- bound MUC1 by an undefined mechanism. Increased levels of sMUC1 have been found in peripheral blood (PB) plasma and serum of patients with adenocarcinomas, which correlate with tumor burden. Assays for the detection of sMUC1 have been cleared by the US Food and Drug Administration (FDA) for use in monitoring disease activity in patients with breast cancer. The function of MUC1 remains to be defined. From an immunologic standpoint, both an immunostimulatory and an immunoinhibitory role have been proposed for membrane- bound MUC1. For sMUC1 , an immunoinhibitory role has been ascribed because sMUC1 inhibits human T-cell proliferation and natural killer cell activity in vitro. Other studies suggest that sMUC1 may be an important immunosuppressive agent in patients with MUC1-bearing malignancies, as evidenced by the poor response to active specific immunotherapy in patients with metastatic adenocarcinomas who have highly elevated sMUC1 levels.

Anti-nuclear antibodies (ANAs, also known as antinuclear factor or ANF) are autoantibodies that bind to contents of the cell nucleus. In normal individuals, the immune system produces antibodies to foreign proteins (antigens) but not to human proteins (autoantigens). There are many subtypes of ANAs such as anti-Ro antibodies, anti-La antibodies, anti-Sm antibodies, anti-nRNP antibodies, anti-Scl-70 antibodies, anti-dsDNA antibodies, anti-histone antibodies, antibodies to nuclear pore complexes, anti-centromere antibodies and anti-sp100 antibodies. Each of these antibody subtypes binds to different proteins or protein complexes within the nucleus. They are found in many disorders including autoimmunity, cancer and infection, with different prevalences of antibodies depending on the condition. This allows the use of ANAs in the diagnosis of some autoimmune disorders, including systemic lupus erythematosus, Sjogren's syndrome, scleroderma, mixed connective tissue disease, polymyositis,

dermatomyositis, autoimmune hepatitis and drug induced lupus. It was not yet measured or detected in diseases which can be correlated to lung cancer. The ANA test detects the auto- antibodies present in an individual's blood serum. The common tests used for detecting and quantifying ANAs are indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA). In immunofluorescence, the level of autoantibodies is reported as a titre. This is the highest dilution of the serum at which autoantibodies are still detectable. Positive

autoantibody titres at a dilution equal to or greater than 1 :160 are usually considered as clinically significant with immune disorders. Positive titres of less than 1:160 are present in up to 20% of the healthy population, especially the elderly. Although positive titres of 1 :160 or higher are strongly associated with autoimmune disorders, they are also found in 5% of healthy individuals. Usually, there is no detectable ANA in the blood (negative test).

Sometimes, however, people who do not have any specific disease may have low levels of ANA in a titer range between 1 :40 and 1 :60 for no obvious reason. The mucin/muc-1 peptide according to the invention is the mature human glycoprotein directed to the muc-1 antigen and comprises the muc-1core repeating peptide unit of the following 20 amino acids:

STAPPAHGVTSAPDTRPAPG TAPPAHGVTSAPDTRPAPGS

APPAHGVTSAPDTRPAPGST

PPAHGVTSAPDTRPAPGSTA

PAHGVTSAPDTRPAPGSTAP

AHGVTSAPDTRPAPGSTAPP HGVTSAPDTRPAPGSTAPPA

GVTSAPDTRPAPGSTAPPAH

VTSAPDTRPAPGSTAPPAHG

TSAPDTRPAPGSTAPPAHGV

SAPDTRPAPGSTAPPAHGVT APDTRPAPGSTAPPAHGVTS

PDTRPAPGSTAPPAHGVTSA

DTRPAPGSTAPPAHGVTSAP

TRPAPGSTAPPAHGVTSAPD

RPAPGSTAPPAHGVTSAPDT PAPGSTAPPAHGVTSAPDTR

APGSTAPPAHGVTSAPDTRP

PGSTAPPAHGVTSAPDTRPA

GSTAPPAHGVTSAPDTRPAP including (i) all biologically active isoforms, variants, mutants and truncated forms thereof including glycosylated, non-glycosylated, partially glycosylated forms, and including forms with modified glycosylation and/or amino acid residue pattern; (ii) any biologically active recombinant or synthetic 20mer peptide consisting of any of the core repeating peptide units as specified above, including peptides with modified amino acid residue pattern; (iii) any biologically active recombinant or synthetic, optionally modified peptide or polypeptide based on one or more of any of the core repeating peptide units as specified above, or any biologically active recombinant or synthetic, optionally modified peptide or polypeptide comprising at least one of said core repeating peptide units and partial sequence tracks of a further repeating unit, including the 25mer peptide having the peptide sequence:

STAPPAHGVTSAPDTRPAPGSTAPP (SEQ ID No. I)

(iv) all lipid forms of (i) to (iii), including STAPPAHGVTSAPDTRPAPGSTAPP-K-palmitoyl-(G), designated as designated as "BLP-25" and including all forms, variants, and derivatives thereof, as described in

WO 1998/50527 and WO 2005/112546,

(v) all proteins, fusion proteins included, comprising the peptide or polypeptide forms as specified above,

(vi) all formulations of human muc-1 , or peptide or polypeptide forms thereof as specified above, preferably any liposomal formulation, and

(vii) all formulations of muc-1 nucleic acids encoding the mature muc-1 protein, and the peptide, polypeptide and lipopetide forms as specified above in combination or association preferably via a liposome with an adjuvant, preferably MPL(3-Odesacyl-4'-monophosphoryl lipid), Lipid A, or low-toxic variants of LPS.

"L-BLP25" or "tecemotide" according to the invention is the combination or mixture of lipopetide BLP-25 or any other peptide sequence as specified above and an adjuvant, preferably MPL or Lipid A, both partners integrated in a liposomal preparation, wherein BLP- 25 (or a similar lipopeptide) and the adjuvant are present in a ratio 1 : 1 up to 5 : 1 by weight, preferably approximately 2 : 1. The BLP-25 lipopeptide provides the antigenic specificity for the T-cell response, while the adjuvant (MPL, Lipid A) enhances the cellular immune responses. The liposomal delivery system is designed to facilitate uptake of the vaccine by antigen-presenting cells (APCs) delivering the lipopeptide into the intracellular space, finally leading to presentation of peptides vial HLA-1 and HLA-II molecules of the HLA complex. This is expected to elicit a muc-1 specific cellular immune response mediated by T-cells, including a CTL response.

It has been found previously by the inventors, and described hereby again, that in a comprehensive statistical analysis of clinical trial EMR63325-001 (START) vaccination with the known muc-1 lipopeptides, preferably L-BLP25/tecemotide, is effective in combination with chemo-radiotherapy if applied to a cohort of lung tumor patients, preferably, patients suffering from non-small cell lung cancer (NSCLC), and most preferably patients suffering from unresectable stage III NSCLC.

According to this invention, there are the following two new findings in detail, based on further detailed statistical and medical subgroup analyses of the START phase III trial EMR63325- 001 :

The first new finding is that L-BLP25/tecemotide is more effective in this setting in patients who have elevated blood concentrations of anti-nuclear antibodies (ANA) at the time just prior to starting the treatment with L-BLP25/tecemotide (from -4 weeks to 0 days prior to start of L- BLP25/tecemotide treatment = baseline). In detail, ANA titers in blood plasma at baseline were tested with an in vitro diagnostic test kit. Concentrations by this test kit were defined as elevated if the ANA-titer was greater than or equal 1 :160. However, it can be seen by detailed analysis that even lower titers, i.e. 1 :80 or 1 :40, 1 :20, and 1 :10 can be associated with the effects described here.

In START, patients in the modified intention to treat population (mITT, n= 239) had an adjusted hazard ratio (HR) for overall survival of 0.88 (95% confidence interval [CI] 0.75- 1.03). A subgroup analysis of patients by baseline ANA concentration was carried out in 1193 patients with available baseline ANA concentration. Patients with ANA titers at baseline of greater than or equal 1 :160 were 128 compared to 1065 who had negative ANA titers

(smaller than 1 :160). Those 128 patients with ANA titers of >1 :160 had a HR in a range between 0.4 and 0.6 in favor of tecemotide treatment (95% CI 0.25-0.69, p=0.0007, Figure 2). Those 1065 patients with ANA titers lower than 1 :160 had a HR in arrange of 0.75 and 0.98 (0.83, 1.17) and no significant or notable treatment effect of L-BLP25/tecemotide (Figure 2). The interaction between ANA titer and treatment effect was statistically notable

(p=0.0016). A more notable treatment effect of L-BLP25 on survival time for elevated baseline ANA titers was also observed within the group of subjects with prior concomitant chemo- radiotherapy (HR=0.27, 95% CI 0.14-0.53, p=0.0001 , Figure 3).

The second new finding resulting from the START trial EMR63325-001 is that L-BLP25 is more effective in patients who have elevated blood concentrations of soluble MUC1 prior to starting treatment with L-BLP25/tecemotide. Soluble MUC1 (sMUC1) was tested in blood plasma using an in vitro diagnostic test kit and baseline sMUC1 concentrations were available from 1097 patients of the mITT population. In particular, in START, high sMUC1

concentrations were associated with a poor prognosis in the placebo arm which is in line with the known negative prognostic effect of this marker. A favorable effect of L-BLP25/tecemotide over placebo (HR<1) was observed in subgroups defined by elevated baseline sMUC1 concentrations. In particular, a favorable and significant treatment effect was seen in subjects with sMUC1 baseline concentrations > 22 lU/mL, preferably between 22 lU/mL and 25 lU/mL (n=399), i.e. concentrations exceeding the upper limit of normal of the employed test kit. Here, the HR for OS between the tecemotide and the placebo group was in a range between

0.60 and 0.70 (95% CI 0.52, 0.89) in favor of tecemotide (Figure 2). In contrast, patients with SMUC1 <25 and preferably < 22 lU/mL (n=698) had a HR of 0.85 - 0.99 (95% CI 0.80, 1.23),

1. e. no treatment benefit from L-BLP25/tecemotide. This effect was even more notable in the small subgroup of subjects with sMUC1 between 25 and 35 lU/mL, preferably > 32 lU/mL (n=199). Here, the HR was between 0.40 and 0.50, preferably around 0.48 (95% CI 0.33, 0.68) in favor of L-BLP25 / tecemotide over placebo (Figure 2). There was a notable interaction between sMUC1≤/>32 lU/mL and treatment (p=0.0002).

Subgroup analyses were also conducted in the subgroup of patients who previously received concurrent chemoradiotherapy (n=806 out of 1239). Here sMUC1 concentrations were available in 704 out of 806 patients and sMUC1 concentrations of >25 lU/mL were present in 256 of the 704 patients. The HR for OS was between 0.50 and 0.60, preferably around 0.55 (95% CI 0.39, 0.78). There was a notable treatment interaction (p=0.0248). sMUC1 concentrations of >32 lU/mL were present in 120 out of 704 patients. The treatment effect of L-BLP25/tecemotide was even more pronounced in this subgroup (HR 0.35 - 0.40, 95% CI 0.25, 0.63, Figure 3).

The data presented according to the invention support that both ANA titers and sMUC1 concentrations in peripheral blood at baseline, i.e. before the start of L-BLP25/tecemotide treatment are potential diagnostic markers to predict the treatment effect of L- BLP25/tecemotide in patients with stage III unresectable NSCLC who previously underwent chemoradiotherapy.

The invention comprises "chemo-radiotherapy". Chemo-radiotherapy according to the invention includes "chemotherapy ". Chemo-radiotherapy also includes "radiotherapy" carried out by radiation according to standard methods or by administration of radio-labelled compounds. According to the invention radiation is preferred. Chemo-radiotherapy according to the invention usually starts with chemotherapy followed by radiotherapy. However, starting therapy with radiotherapy is also applicable. Chemotherapy is carried out by administration of at least one "chemotherapeutic agent", preferably a platinum-based drug, such as cisplatin or carboplatin. According to the invention the platinum- based chemotherapeutic agents are administered daily, weekly or every 2 to 5 weeks, dependent on the dose duration and number of administrations.

Chemotherapy according to the invention comprises administration of chemotherapeutic agents which are according to the understanding of this invention a member of the class of cytotoxic agents, and include chemical agents that exert anti-neoplastic effects, i.e., prevent the development, maturation, or spread of neoplastic cells, directly on the tumor cell, and not indirectly through mechanisms such as biological response modification.

Preferred chemotherapeutic agents according to the invention which are administered in the chemo-radiotherapy settings of the invention are platinum-based agents, such as cisplatin or carboplatin. However, other chemotherapeutic agents as specified below, may be also used. In addition further chemotherapeutic agents or other anti-cancer agents can be administered to improve efficacy of the claimed therapy. There are large numbers of anti-neoplastic agents available in commercial use, in clinical evaluation and in pre-clinical development, which could be included in the present invention for treatment of tumors / neoplasia by combination therapy. It should be pointed out that the chemotherapeutic agents can be administered optionally together with above-said antibody drug. Examples of chemotherapeutic or agents include alkylating agents, for example, nitrogen mustards, ethyleneimine compounds, alkyl sulphonates and other compounds with an alkylating action such as nitrosoureas, cisplatin and dacarbazine; antimetabolites, for example, folic acid, purine or pyrimidine antagonists; mitotic inhibitors, for example, vinca alkaloids and derivatives of podophyllotoxin; cytotoxic antibiotics and camptothecin derivatives. Preferred chemotherapeutic agents or

chemotherapy include amifostine (ethyol), cabazitaxel, cisplatin, dacarbazine (DTIC), dactinomycin, docetaxel, mechlorethamine, streptozocin, cyclophosphamide, carrnustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), doxorubicin lipo (doxil), gemcitabine (gemzar), daunorubicin, daunorubicin lipo (daunoxome), procarbazine, ketokonazole, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11 , 10-hydroxy-7-ethyl-camptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil and combinations thereof.

In a preferred embodiment of the invention a liposomal formulation is provided, wherein the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin or carboplatin, and the non-platinum based chemotherapeutic agent is selected from the group consisting of vinorelbine, etoposide, paclitaxel, docetaxel, vindesine, gemcitabine, ifosfamide and pemetrexed.

In another patient setting the chemotherapy includes administering cyclophosphamide (CPA). CPA treatment can create a favorable regulatory environment for vaccination by inhibiting regulatory T cells (T reg ) and promoting antitumor immune responses through direct cytotoxic effects on the tumor mass, which can be enhanced by tecemotide vaccination. Low-dose CPA has been shown to enhance the antitumor effects of tecemotide in a human MUC1 transgenic (hMUC1 g) lung cancer model when given 3 days before treatment initiation. 1 In a phase III clinical trial of non-small cell lung cancer (NSCLC), CPA plus tecemotide does not meet the primary endpoint of prolonging overall survival; however, pre-defined subgroup analyses revealed a clinically meaningful prolongation of OS in patients previously treated with concurrent chemo/RT (HR 0.65 - 0.80, 95% CI 0.64-0.95, p=0.016).

Instead of chemotherapeutic agents, administration of immunotherapeutic agents are favorable according to the invention in addition to said platinum-based chemotherapeutic agents. Suitable immunotherapeutic agents according to the invention are, for example, anticancer antibodies, such as anti-VEGF(R) antibodies or anti EGFR antibodies.

In more detail, a platinum-based chemotherapeutic agent, like cisplatin and carboplatin can be combined according to the invention with drugs such as: taxanes, like pacitaxel and docetaxel; anti-angiogenic molecules such as bevacizumab, anti-metabolic agents such pemetrexed and gemcitabine; topo-isomerase inhibitors such as etoposide or irinotecan, vinca alkaloids such as vinorelbine and vinblastine, EGFR targeting agents such as cetuximab, panitumumab, erlotinib, gefitinib and afatinib, and alkylating agents such as ifosfamide. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells by causing destruction of cells. The term is intended to include radioactive isotopes, chemotherapeutic agents, immunotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. The term may include also members of the cytokine family, preferably IFNy as well as anti- neoplastic agents having also cytotoxic activity.

The term "anti-cancer agent" describes all agents which are effective in cancer therapy. The term includes, cytotoxic agents, chemotherapeutic agents, and immunotherapeutic agents.

The term "concurrent or concomitant chemo-radiotherapy" means according to the invention a combination of chemotherapy and radiotherapy which are timely at least overlapping, preferably overlapping by at least 10% - 15% calculated from the duration of the respective chemotherapy. Preferably chemo- and radiotherapy are overlapping more than 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%. A preferred overlapping range is between 10% - 100%, preferably 20 - 100%, more preferably 70 - 100%, most preferably 50 - 100%. In the most preferred embodiment, radiotherapy is started after starting chemotherapy and is completed after completion of chemotherapy (100% overlap). The values indicated refer to a single patient. They may vary in a statistical consideration of a cohort of patients. The terms

"concurrent" and "concomitant" are used synonymously in this document. The term "sequential chemo-radiotherapy" means according to the invention a combination of chemotherapy and radiotherapy which are timely not overlapping at all or are overlapping by less than 10%, more preferably less than 5%, most preferably less than 1 % calculated from the duration of the respective chemotherapy. In the sequential chemo-radiotherapy setting according to the invention, which is not overlapping at all, radiotherapy treatment starts preferably 1 - 28 days, more preferably 1 - 21 , most preferably 7 - 14 days after completion of radiotherapy. The values indicated refer to a single patient. They may vary in a statistical consideration of a cohort of patients.

According to the invention the chemo-radiotherapy is applied and completed before the vaccination with said liposomal formulation is started.

Chemotherapy is applied according to the invention by at least two cycles, preferably 2 - 8 cycles, more preferably 2 - 5 cycles. One cycle is between 21 and 35 days, preferably between 21 - 28 days. The dose regimen of the chemotherapeutic agent, preferably the platinum-based agents is dependent on various possible patient- and drug-related conditions and properties. Usually, cisplatin is applied in doses varying from 50 - 120 mg / m 2 and per cycle. Carboplatin may be applied according to the invention in doses of 500 - 1500 mg per single dose and per cycle.

Radiotherapy is carried out according to the invention - as mentioned - by standard radiation, wherein a total of 40 - 120 Gy are applied, preferably at least 50 Gy, more preferably between 50 and 75 Gy. The radiation therapy is usually fractionated, wherein 1.5 - 3.5 Gy are applied per day for at least four days, preferably 5 - 7 days in sequence. The total radiation dose is to be applied according to the invention within 21 - 35 days, preferably within 28 days. If necessary or favourable, boost doses of 3.5 - 15 Gy, preferably 5 - 10 Gy can be applied at the beginning of radiation or in an intermediate interval. According to the invention vaccination is applied after completion of the chemo-radiotherapy. The liposomal formulation comprising the lipopeptide of the invention is applied 7 - 35, preferably 14 - 28 days after completion of said radiotherapy. It could be shown that the efficacy of the vaccination treatment after chemo-radiotherapy is not influenced negatively if vaccination is not started later than 84 - 98 days. Vaccination is applied according to the invention during the initial phase every 5 th - 9 th , preferably every 7 th day. The initial phase is completed after 6 - 8 weeks after start.

Thereafter, every 5 - 7 weeks, preferably every 6 weeks a further vaccination dose is applied according to the invention. One single dose of the liposomal formulation should contain according to the invention 500 - 1.200 pg of said lipopeptide, preferably 700 - 900 pg.

The chemo-radiotherapy vaccination treatment can be accompanied by administration of an agents that is capable to modulate the immune system. By, for example, applying a relatively low dose of cyclophosphamide between 100 - 400 mg/m preferably 250 mg/m 2 the immune system of the patient can be activated or enhanced. Usually, a single dose before start of the vaccination, as a rule 1 to 5 days, preferably 2 - 5 days, should be sufficient to be effective.

Overall survival (OS)" means a term that denotes the chances of staying alive for a group of individuals suffering from a cancer. It denotes the percentage of individuals in the group who are likely to be alive after a particular duration of time. At a basic level, the overall survival is representative of cure rates.

"Hazard rate (HR)" means according to the invention a measure of how often a particular event happens in one group compared to how often it happens in another group, over time. In cancer research, hazard ratios are often used in clinical trials to measure survival at any point in time in a group of patients who have been given a specific treatment compared to a control group given another treatment or a placebo. A hazard ratio of one means that there is no difference in survival between the two groups. A hazard ratio of greater than one or less than one means that survival was better in one of the groups.

"Clinical/therapeutic response" or "responsiveness" means a response to drug intake that can be detected and appreciated by change in signs and symptoms caused by the disease for which the drug, or whatever kind of therapy, is being taken.

SHORT DESCRIPTION OF THE FIGURES:

Fig. 1 : Study design of EMR 63325-001 (tecetomide/L-BLP25) ("START")

Fig. 2: Subgroup analysis of overall survival in study EMR63325-001 ("START") by baseline markers anti-nuclear antibodies and soluble MUC1 in patients of the mITT (n=1239). Fig. 3: Subgroup analysis of overall survival in study EMR63325-001 (START) by baseline markers anti-nuclear antibodies and soluble MUC1 in patients of the mITT who previously were treated with concurrent chemoradiotherapy (n=806).

Fig. 4: Progression-free survival (PFS) and Time to treatment failure (TTF): primary analysis population

Fig.5: Progression-free survival: concurrent vs sequential chemo-radiotherapy

Fig. 6: Progression-free survival and TTF: primary analysis population

Fig. 7: Exploratory biomarker analysis: rationale

Fig. 8: Overall survival by HLA subgroups Fig. 9: Overall survival by lymphocytes, NLR, sMUC1 and ANA: mITT

Fig. 10: Overall survival by lymphocytes, NLR, sMUC1 and ANA: concurrent vs sequential chemo-radiotherapy.

Fig. 11 : Biomarker analysis: HLA type and overall survival

Fig. 12: Biomarker analysis: overall survival by baseline ANAs, lymphocyte count, NLR sMUC1.

EXAMPLE 1 :

L-BLP25 is a MUC1 antigen specific cancer immunotherapy. Here, the results report results from the phase III START study of L-BLP25 in patients (pts) not progressing after primary chemoradiotherapy (CRT) for stage III NSCLC. This following summarizes the key results of the 100% events analysis of the START trial. All analyses are based on a dataset with clinical cut-off date of 08 August 2012. The study design is depicted in Fig. 1.

The design and objectives of this trial are described in the clinical trial protocol and in the statistical analysis plan (SAP) V 2.0. In brief, subjects with unresectable stage III NSCLC who have demonstrated either stable disease or objective response following primary chemoradiotherapy (concomitant or sequential) were randomized 2:1 either to cyclophosphamide and L-BLP25 (investigational group) or to placebo (control group), respectively, in a double- blinded fashion. The randomization was stratified by disease stage (stage IIIA or IIIB), response to primary chemo-radiotherapy (stable disease or objective response), type of primary chemo-radiotherapy (concomitant or sequential), and region (1 : North America

[Canada, US] and Australia, 2: Western Europe, or 3: ROW [Mexico, Central and South America, Eastern Europe and Asia]). The purpose to select these stratification factors was related to prognostic factors in stage ill NSCLC). Subjects in both treatment groups in addition received best supportive care according to the investigator's discretion. The primary variable of this trial was survival duration. The trial was powered with 90% to detect a significant HR of 0.77 at significance level alpha 0.05 (2-sided) assuming a median survival of 20 months in the control group.

The protocol was amended to modify the primary analysis population, which is in principle based on the intention-to-treat (ITT) population (n=1513) but under prospective exclusion of all subjects randomized during the 6 months (= 26 weeks) period prior to the clinical hold (n=274). These subjects were excluded regardless of the actual survival outcome (modified ITT or mITT population). The rationale for this change was the assumption that an

uninterrupted initial treatment with L-BLP25 of at least 6 months would produce a clinically relevant effect. The modified ITT (mITT) as primary analysis population and the SAP V2.0 was agreed upon with the FDA under a Special Protocol Assessment agreement, and was considered to be acceptable by the MEB, MHRA, MPA and the PEI (HAs of the Netherlands, UK, Sweden and Germany, respectively) in the context of Scientific Advice procedures. Between initiation of screening in January 2007 and end of recruitment on

15 November 2011 , 1908 subjects were screened and 1513 were randomized (ITT

population) to the L-BLP25 active treatment group (n= 006, 66.5%) or to the placebo treatment group (n=507, 33.5%). The safety population consists of a total of 1501 subjects with 1024 subjects in the L-BLP25 group and 477 subjects in the placebo group. The difference of 12 subjects between the ITT and the safety population reflects subjects who had been randomized but who had not started treatment. Of note, 24 subjects in the safety analysis set who had been randomized to the placebo group but received at least one administration of cyclophosphamide or L-BLP25 (major protocol violation) were evaluated in the active treatment group. Also, the placebo group of the safety analysis set contains 1 subject originally randomized to the L-BLP25 treatment group who received a saline pre- infusion only.

From Jan 2007 to Nov 201 , 15 3 pts with unresectable stage III NSCLC that did not progress after CRT (platinum based chemo and >50 Gy) were randomized (2:1 ; double-blind) to L-BLP25 (806 pg lipopeptide) or placebo (PBO) SC weekly x 8 then Q6 weeks until disease progression or withdrawal. Cyclophosphamide 300 mg/m 2 x 1 or saline was given 3 days prior to first L-BLP25/PBO dose. Primary endpoint was overall survival (OS).

The primary analysis population (n=1239) was defined prospectively to try to account for a clinical hold by excluding pts randomized 6 months (m) before the hold. Arms were balanced for baseline characteristics. Median age was 61 y; 38.2% had stage IIIA and 61.3% IIIB; 65% had concurrent and 35% sequential CRT. Median OS was 25.6 m with L-BLP25 vs. 22.3 m with PBO (adjusted HR 0.88, 95% CI 0.75-1.03, p=0.123). Secondary endpoints time-to- progression and time-to-symptom-progression support consistency of results: HR 0.87 (95% CI 0.75-1.00, p=0.053) and 0.85 (95% CI 0.73-0.98, p=0.023). In predefined subgroup analyses, pts with concurrent CRT (n=806) had median OS of 30.8 m (L-BLP25) vs. 20.6 m (PBO; HR 0.78, 95% CI 0.64-0.95, p=0.016), while median OS with sequential CRT was 19.4 m (L-BLP25) vs. 24.6 m (PBO; HR 1.12, 95% CI 0.87-1.44, p=0.38; interaction p=0.032, Cox PH model). Sensitivity analyses revealed that there was no OS benefit in pts randomized 6 m before the hold (HR 1.09, CI 0.75-1.56, p=0.663). L-BLP25 was well tolerated with no safety concerns identified and no emergent evidence of immune related adverse events. L-BLP25 maintenance therapy in stage III NSCLC was well tolerated, but did not significantly prolong OS. Sensitivity analyses showed a smaller treatment effect due to the clinical hold, suggesting that longer uninterrupted treatment with L-BLP25 is required. Clinically meaningful prolongation of OS was observed in the predefined subgroup of pts with primary concurrent CRT. Out of the 1024 subjects treated in the L-BLP25 group 6 subjects discontinued treatment after the initial cyclophosphamide infusion and 1018 subjects were treated further. In the placebo group 6 out of 477 subjects discontinued treatment after the initial saline infusion and 471 subjects were treated with placebo. Median duration of treatment was 32.4 weeks in the L-BLP25 group and 26.6 weeks in the placebo group (safety analysis set).

Median number of vaccinations administered was 1 both in the L-BLP25 group and in the placebo group (safety analysis set).

The primary objective of this trial, i.e. to demonstrate a statistically significant prolongation of overall survival with L-BLP25 treatment assuming a true HR of 0.77 in the population under study, was not met. The Forest plot shows overall survival results for predefined baseline characteristics and randomization strata, respectively, in the mITT population. These baseline characteristics and randomization strata were defined a priori because of the known or assumed prognostic impact on survival time of NSCLC patients. For each of the illustrated baseline characteristics and randomization factors the HR estimate including 95% CI is displayed (for the

randomization strata an unstratified Cox model with treatment as single factor was used). The HR estimate is depicted by a filled circle and the size of the circle is proportional to the subgroup sample size.

In nearly all subgroups a favorable effect of L-BLP25 over placebo (HR<1) was observed except for tumor histology adenocarcinoma, and sequential chemo-radiotherapy). In small subgroups like Asian / Pacific Islander and Latino / Hispanic or Never Smokers a treatment effect in favor of placebo was seen in addition (HR > 1) but these groups are too small for a meaningful interpretation. The most prominent subgroups consist of the prospectively defined randomization stratum differentiating prior concomitant chemo-radiotherapy and sequential chemo-radiotherapy. The concomitantly pretreated subgroup with 806 out of 1239 subjects (65%) showed a positive effect from L-BLP25 treatment (mOS 30.8 vs. 20.6 months, adjusted HR 0.78, [95% CI 0.64-0.95], p=0.016), whereas in the sequentially pretreated subgroup this favorable effect of L-BLP25 was not observed and placebo seemed to be more favorable although the confidence interval of the HR covered unity (mOS 19.4 vs. 24.6, adjusted HR 1.12, [95% CI 0.87-1.44]). Time to Progression by prior chemo-radiotherapy: Analyses of TTP were repeated in the stratum of prior chemo-radiotherapy. In the stratum of subjects with prior concomitant chemo- radiotherapy a HR of 0.85 was observed ([95% CI 0.71-1.02], p=0.078 not adjusted for multiplicity). In contrast, in subjects with prior sequential chemo-radiotherapy the treatment effect in favor of L-BLP25 was less clear (HR 0.91 , [95% CI 0.72-1.15], p=0.437 not adjusted for multiplicity). These observations were in line with the observations made for overall survival. All analyses were repeated for these two prominent randomization strata of concomitant/concurrent (cone) versus sequential (seq) chemo-radiotherapy in the frame of post-hoc analyses. As shown for all predefined subgroups of sufficient size in the concomitant stratum a benefit in favor of L-BLP25 treatment was observed. Results for predefined subgroups in the sequential stratum were observed to be more heterogeneous. Of note, for subjects in the sequential subgroup who were either female or had adenocarcinoma, a HR in favor of placebo was observed in the subgroup of sequential chemo-radiotherapy. A detrimental effect for subjects in these subgroups could not be excluded and affected subjects on ongoing treatment were informed and re-consented.

Subgroup analyses by randomization strata and other pre-defined subsets revealed a treatment benefit in favor of L-BLP25 in subjects previously treated with concomitant chemo- radiotherapy, whereas in sequentially pretreated subjects a trend for longer survival times were observed in the placebo group. In detail, in the subgroup of subjects previously treated with concomitant chemo-radiotherapy a HR of 0.78 with [95% CI 0.64-0.95] was observed (n=806 out of 1239, mOS 30.8 vs. 20.6 months, p=0.016 two-sided). In contrast, the subgroup of subjects with prior sequential chemo-radiotherapy showed a HR of 1.12 with a [95% CI 0.87-1.44], n=433 out of 1239, mOS 19.4 vs. 24.6 months, p=0.38 two-sided).

Importantly, for the subgroup of concomitantly pre-treated subjects, further subgroup analyses within this stratum revealed similar effects in favor of L-BLP25 treatment. In contrast, results for predefined subgroups in the sequential stratum were observed to be more heterogeneous. Secondary endpoints showed HRs <1.0 (mTTSP 14.2 vs. 11.4 months, respectively, HR 0.85, p=0.023 two sided, [95% CI 0.73-0.98]; TTP 10.0 vs. 8.4 months, respectively, HR 0.87, p=0.053 two sided, [95% CI 0.75-1.00] and PFS 9.6 vs 7.7 months, HR 0.87, p=0.0359, [95% CI 0.76-0.990]). Similarly to the subgroup analyses for the primary endpoint, subjects with prior concomitant chemoradiotherapy had a tendency towards a treatment effect in favor of L-BLP25 treatment in TTSP and TTP which was more pronounced than in the sequential stratum. Although not statistically significant, a treatment effect in favor of L-BLP25 in the overall primary analysis population was observed in the primary and secondary endpoints (OS, TTSP, TTP, respectively). This observed treatment effect was more pronounced in subjects within the stratum of prior concomitant chemo-radiotherapy. The observed effect in this subgroup was clinically meaningful (HR 0.78, [95% CI 0.64-0.95], mOS 30.8 vs. 20.6 months, p=0.016). Such effect was not observed in the subgroup of subjects with prior sequential chemo-radiotherapy (HR > 1). Sensitivity analyses support the possibility that the observed treatment benefit for L-BLP25 in the concomitant stratum may have been underestimated due to the impact of the clinical hold of the trial in 2010.

EXAMPLE 2:

Testing for ANAs and MUC1 : tests are well known in the art, such as:

The detection and semi-quantitation of autoantibodies aid in the diagnosis of autoimmune diseases (1). The Kallestad HEp-2 Test detects autoantibodies to nuclear (ANA) antigens. Laboratories have used indirect fluorescent antibody (IFA) procedures to detect

autoantibodies since 1957. In these procedures, a fluorescent antibody serves as a marker for an antigen-antibody binding reaction which occurs on a substrate surface. Among the substrates frequently used in the IFA procedure are human epithelial (HEp-2) cells. Other common substrates, such as mouse kidney and stomach, and Crithidia luciliae (a

hemoflagellate), are also available from Bio-Rad Laboratories. Observation of a specific pattern of fluorescence on the substrate indicates the presence of autoantibodies in the patient's serum. A positive ANA result usually occurs in patients with autoimmune disorders such as systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), Sjogren's syndrome (SS), and ogressive systemic sclerosis (PSS) (2). However, the incidence of low-titer ANA positives increases with age in normal individuals (3). Therefore, the presence of low-titer antibody should be interpreted in the context of other clinical information. Autoantibodies in a test sample bind to antigens in the substrate.

Washing removes excess serum from the substrate. Fluorescein conjugated (FITC) antiserum added to the substrate attaches to the bound autoantibody. After a second washing step to remove excess conjugate, the substrate is coverslipped and viewed for fluorescent patterns with a fluorescent microscope. Observation of a specific fluorescent pattern(s) on the substrate indicates the presence of autoantibodies in the test sample.

Further details of ANA testing is described and summarized by Fernandez-Madrid et al., Vol. 5, 1393-1400, June 1999 Clinical Cancer Research 1393: "Antinuclear Antibodies as

Potential Markers of Lung Cancer.

A further general sandwich based test principle is: Total duration of assay: 18 minutes.1st incubation: 20 ~L of sample are automatically prediluted 1 :10 with Elecsys Diluent Universal. The antigen (in 20 ul of prediluted sample), a biotinylated monoclonal CA 15-3-specific antibody, and a monoclonal CA 15-3-specif ic antibody labeled with a ruthenium complex react to form a sandwich complex. 2nd incubation: After addition of streptavidin-coated microparticles, the complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the

microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M.

Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode. sMUC1 levels can be determined by standard methods (e.g.: Ishikawa et al.; 2008; Int. J. Cancer: 122, 2612-2620)