WO2013076222A9

WO2013076222A9 - Differential annexin a3 measurements of serum and blood derivatives or fractions thereof for the diagnosis of prostate cancer

Info

Publication number: WO2013076222A9
Application number: PCT/EP2012/073417
Authority: WO
Inventors: André SCHRATTENHOLZ; Gerhard Schwall; Slobodan Poznanovic; Karlfried GROEBE
Original assignee: Proteosys Ag
Priority date: 2011-11-23
Filing date: 2012-11-22
Publication date: 2013-08-01
Also published as: WO2013076222A1

Abstract

The present application refers to the measurement of Annexin A3 in body fluids, particularly serum, plasma and/or blood, for the in vitro diagnosis of cancer, particularly of prostate cancer.

Description

Differential Annexin A3 measurements of serum and blood derivatives or fractions thereof for the diagnosis of prostate cancer

Description

Annexin A3 (ANXA3) has been described as a biomarker for prostatic disease previously (Kollermann et al., 2008; Schostak et al., 2009). WO 2005/078124 discloses that Annexin A3 is a suitable marker for the diagnosis of cancer, particularly prostate cancer sub-types. WO 2006/125580 discloses the use of Annexin A3 for the diagnosis of prostate cancer. WO 2007/141043 describes a separate measurement of extracellular and of intracellular Annexin A3 in a sample. WO 2008/125262 describes the determination of the presence, amount and/or distribution, particularly the intracellular distribution/localization, of Annexin A3 in a sample. WO 2010/034825 describes antibodies recognizing native Annexin A3 and the use for diagnostic and therapeutic applications. The contents of these documents are herein incorporated by reference.

Co-pending application US 61 , 563, 123 describes measuring two distinct conformations of Annexin A3 in body fluid samples by using two antibodies specifically directed against one of these distinct conformations. The content of this document is herein incorporated by reference.

A subject-matter of the invention is a method for the in vitro diagnosis of cancer, particularly prostate cancer, comprising at least one measurement of a specific conformation of human Annexin A3 in a body fluid, wherein said specific conformation of human Annexin A3 is selected from a first conformation which is predominantly present in a first type of vesicles, particularly in prostasomes, and a second conformation which is predominantly present in a second type of vesicles, particularly in vesicles comprising phospholipid-based membranes, and wherein the first type of vesicles is different from the second type. Particularly, the first and the second conformation are present in extracellular vesicles.

The method of the invention is particularly suitable for the diagnosis of an epithelial cancer, more particularly for the diagnosis of prostate cancer. The conformation-specific measurement allows an improved diagnostic performance of an ANXA3-based test since the individual conformations of ANXA3 are differently associated with cancer or other disorders, respectively.

More particularly, the inventors have found that one of the different conformations of human Annexin A3 is directly associated with prostate cancer, i.e. an increased amount of the first conformation of human Annexin A3 is associated with prostate cancer, whereas the other conformation of human Annexin A3 is inversely associated with prostate cancer, i.e. an increased amount of this conformation compared to a control is associated with benign hyperplasia and/or absence of cancer.

The two distinct conformations of Annexin A3 preferably relate to ANXA3 present in prostasomes (small vesicles with detergent-resistant, cholesterol- rich membranes (Duijvesz et al., 201 1 ; Tavoosidana et al., 201 1 ) and ANXA3 present in normal phospholipid membranes.

The conformation-specific measurement is carried out in a body fluid sample. The body fluid sample is a sample of a human body fluid, preferably from a subject in need for the diagnosis of cancer. This includes a subject in whom cancer has not yet been diagnosed, and a subject in whom cancer has been diagnosed and the course and/or progression of the disease is monitored.

The body fluid is preferably selected from the group consisting of urine, exprimate urine, blood, serum, plasma and further blood derivatives, or seminal fluid, e.g. seminal plasma. More preferably the body fluid is selected from blood serum, plasma and further blood derivatives.

The method of the invention comprises at least one conformation-specific measurement of ANXA3, particularly of extracellular ANXA3. For example, the method may comprise (i) a conformation-specific measurement of the first conformation, or (ii) a conformation-specific measurement of the second conformation, or (iii) conformation-specific measurements of both the first and the second conformation. Preferably, the method of the invention comprises separate measurements of two different conformations of extracellular human Annexin A3, particularly in two different types of human Annexin A3 containing extracellular vesicles which are distinguishable by physical and/or chemical parameters such as calcium dependency, solvent resistance, detergent-resistance and/or density.

A conformation-specific measurement of an individual conformation of human Annexin A3 may be carried out after chemical and/or physical separation of the first and second conformation. Preferably, separation of the two different Annexin A3 conformations is carried out by chemical procedures, e.g. addition of additives such as metal ions, e.g. calcium, organic solvents, e.g. DMSO, DMF, ethanol, isopropanol etc., detergents and/or stabilizers, and/or by physical procedures, e.g. centrifugation, freeze- thaw cycles and/or heating. Preferably, separation involves a chemical procedure, comprising addition of calcium ions in a concentration of e.g. 0.2- 10 mM, preferably 1 -5 mM, and/or addition of an organic solvent such as DMSO in a concentration of 0.5-15% (vol/vol), preferably 1 -10% (vol/vol), to a body fluid, e.g. serum sample, and subsequent physical separation, e.g. by centrifugation. More preferably, the chemical procedure involves addition of calcium ions and solvent.

The inventors have found that the ANXA3 conformation which is associated with prostate cancer precipitates in the presence of 1-5 mM calcium and 1 - 10% DMSO and can thus be centrifuged (e.g. 5,000-12,000 rpm Eppendorf table centrifuge) from serum samples of patients supplemented with said calcium and/or DMSO concentrations, whereas, the ANXA3 conformation associated with benign prostatic hyperplasia remains in solution under these conditions and cannot be centrifuged.

Thus, the conformation-specific measurement may be carried out in a processed sample, e.g. a sample supplemented with metal ions, particularly calcium, and/or an organic solvent, particularly DMSO. Preferably, the sample has been subjected to a physical separation procedure, particularly centrifugation.

Preferably, the method of the invention involves a separate quantitative measurement of the first and/or the second conformation of human Annexin A3. The separate quantification of the two different diagnostic ANXA3 conformations leads to a significant improvement in the diagnostic performance.

The method of the invention requires a conformation-specific measurement of at least one and preferably both different conformations of human Annexin A3. In some embodiments, the conformation-specific measurement may be carried out e.g. in two fractions of a single sample or in two processed, particularly differently processed, sample aliquots. In other embodiments, the separate measurements may be carried out in a single sample without different processing using a conformation-specific detection procedure.

The method of the invention preferably involves the use of a suitable immunological detection procedure, wherein the first and/or the second conformations of human Annexin A3 are determined in conformation-specific measurements by reaction with an antibody which specifically recognises human Annexin A3. The conformation-specific measurement may involve measurement of a processed sample which comprises only one conformation of ANXA3 in a detectable form. In this embodiment, a conformation- unspecific detection reagent, e.g. an antibody may be used. In another embodiment, a conformation-specific detection reagent, e.g. an antibody, may be used. More preferably, the detection reagent is an antibody, e.g. a monoclonal antibody, e.g. an antibody which is described in WO 2007/141043 or in WO 2010/034825, the contents of which are herein incorporated by reference, or a polyclonal antiserum. Specific examples of suitable monoclonal antibodies are antibodies produced by the hybridoma cell lines tgc 5 (DSM ACC 2778), tgc 6 (DSM ACC 2779), tgc 7 (DSM ACC 2780), tgc 8 (DSM ACC 2781 ) or antibodies which bind to the same epitopes on Annexin A3. The indicated hybridoma cell lines have been deposited on 5 May 2006 at DSMZ (Deutsche Sammlung von

Mikroorganismen und Zellkulturen GmbH, Inhoffenstrafte 7b, D-38124 Braunschweig), according to the Budapest Treaty. Further, specific examples of suitable monoclonal antibodies are antibodies produced by the hybridoma cell lines tgc 42 (DSM ACC2972), tgc 43 (DSM ACC 2970), tgc 44 (DSM ACC 2976), tgc 45 (DSM ACC 2974), tgc 46 (DSM ACC 2975), tgc 47 (DSM ACC 2977), tgc 48 (DSM ACC 2971 ) and tgc 49 (DSM ACC 2973), or antibodies which bind to the same epitopes on Annexin A3. The indicated hybridoma cell lines were deposited under the conditions of the Budapest Treaty at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7b, 38124 Braunschweig, Germany) on 17 September 2008. Alternatively, the detection antibody may be a polyclonal antiserum, e.g. the polyclonal antiserum Petros (Wozny et al., 2007).

In certain embodiments of the present invention, the conformation-specific measurement of the first and/or second conformation of human Annexin A3 may involve the use of a first and/or a second antibody, wherein the first antibody recognises the first conformation of human Annexin A3 and the second antibody recognises the second conformation of human Annexin A3. In this embodiment, the two conformations may be determined in a single sample or in two aliquots resulting from a single sample. The first and the second antibody each specifically recognize a different conformation of human Annexin A3 and are capable of distinguishing different conformations thereof. Preferably, one of the conformation-specific antibodies predominantly reacts with a conformation of human Annexin A3 which is associated with prostate cancer, and one of the conformation- specific antibodies predominantly reacts with a conformation of human Annexin A3 which is inversely associated with prostate cancer, i.e. which is associated with benign hyperplasia and/or absence of cancer.

In a preferred embodiment, one of the conformation-specific antibodies is (i) antibody tgc 43 (DSM ACC 2970), (ii) an antibody recognizing the same conformational domain on human Annexin A3 or (iii) antigen-binding fragment of (i) or (ii). Such an antibody recognizes a conformation of ANXA3 which is associated with cancer.

In a further preferred embodiment, one of the antibodies is (i) antibody tgc 44 (DSM ACC 2976), (ii) an antibody recognizing the same conformational domain on human Annexin A3 or (iii) antigen-binding fragment of (i) or (ii). Such an antibody recognizes a conformation of ANXA3 which is associated with absence of cancer and/or benign hyperplasia.

In a preferred embodiment, the immunological detection procedure is a sandwich immunoassay wherein the presence and/or quantity of different conformations of human Annexin A3 in the sample is detected by forming a detectable complex between a capture antibody, Annexin A3 and a detection antibody.

In some embodiments, a first and a second conformation-specific antibody are used as capture antibodies. In other embodiments, which involve measurements in fractionated and/or processed samples, a single, e.g. conformation-unspecific antibody may be used as capture antibody. The capture antibodies are preferably bound to a solid phase, e.g. the wall of a reaction vessel or a particular solid phase, e.g. a nanoparticle. Alternatively, capture antibodies may comprise a solid phase-binding group which allows binding to a solid phase having a complementary binding group (e.g. biotin- streptavidin).

The binding of human Annexin A3 in the sample to the capture antibodies may be determined by using detection antibodies, i.e. further antibodies recognizing human Annexin A3 which carry a detection group (e.g. an optically detectable group such as a fluorescent group) or which can be detected by reaction to a further detection reagent which carries a detection group. Detection antibodies are preferably selected which do not interfere with the binding of the capture antibodies to human Annexin A3. The detection antibody may be a monoclonal antibody, e.g. an antibody described in WO 2007/141043 and WO 2010/034825 (except tgc 43 or tgc 44), the contents of which are herein incorporated by reference. Alternatively, the detection antibody may be a polyclonal antiserum, e.g. the polyclonal antiserum Petros (Wozny et al., 2007).

In order to obtain a quantitative measurement, a calibration may be carried out. Calibration of the measurements may comprise obtaining mathematical derivatives of the concentration measurements, such as a quotient, product, logarithm etc. and/or using additional other antibodies against human Annexin A3, which recognize further conformations of the protein.

Further, it is preferred that the two concentrations of Annexin A3 obtained from the different measurements of individual conformations are calibrated by at least one additional biophysical or biochemical parameter of the corresponding body fluid.

Preferably, the additional biophysical or biochemical parameter is selected from the group consisting of density, viscosity, inflammatory markers, ions, in particular calcium ions, phospholipids, cholesterol, sphingolipids, and enzymes. The diagnostic performance may be improved by combining the two concentrations of Annexin A3 in serum with at least one further cancer marker from other sources of the same patient, such as a tissue sample, in particular a biopsy.

The further cancer marker may be selected from prostate cancer specific markers, e.g. from the group comprising prostate specific antigen (PSA) and variants thereof, PCA3, PCADM-1 , and EPCA-2.

For example, the measurement of Annexin A3 may be combined with measuring prostate specific antigen (PSA), free PSA, % free PSA, complexed PSA (cPSA), and/or other variants of PSA.

Thus, the present invention provides an improved method for the measurement of human Annexin A3 by separately measuring a first conformation of human Annexin A3 and a second conformation of human Annexin A3 in a body fluid sample, wherein an increased amount of Annexin A3 in the first conformation compared to a control is indicative of cancer, particularly prostate cancer, and an increased amount of Annexin A3 in the second conformation is indicative of the absence of cancer, particularly prostate cancer.

Further, the present invention provides a reagent kit for the measurement of human Annexin A3 in a body fluid sample, comprising a first and a second antibody, wherein the first antibody specifically recognizes a first conformation of human Annexin A3 and the second antibody specifically recognizes a second conformation of human Annexin A3.

Furthermore, the present invention provides a reagent kit for the measurement of human Annexin A3 in a body fluid sample, comprising an antibody specifically recognizing human Annexin A3, and a reagent or reagent combination for separating a first conformation of human Annexin A3 from a second conformation of human Annexin A3 in said body fluid sample.

Preferred embodiments of the reagent kit are as described above with regard to the diagnosis methods.

Further, the present invention shall be explained in more detail by the following experimental description:

Figure 1 shows that the antibody tgc 43 (DSM ACC 2970) has a positive correlation to prostate cancer in serum, i.e. ANXA3-values are high in serum of patients with cancer and low in those with benign hyperplasia. The AUROC for 100 patients is 0.739 without further calibration.

The antibody tgc 44 (DSM ACC 2976) however detects another conformation of ANXA3 with an AUROC of 0.638 and an inverse relationship to prostate cancer, i.e. ANXA3-values are high for patients with benign hyperplasia and low for cancer (Fig. 2). The two series of measurements are not correlated to each other: As shown in Figure 3, the Spearman correlation of R²= 0.0409 clearly demonstrates that although both antibodies bind to similar domains on ANXA3 and recognize the protein with high specificity and no cross- reactivity, they obviously distinguish two different conformations.

In contrast to previous measurements where only one of these two conformations was targeted (namely the one with inverse relationship to cancer like tgc 44) with one detection antibody, here we see, quite surprisingly, that that combination of the two capture anti-ANXA3 antibodies substantially improves the performance. As shown in Figure 4, a linear combination of the measurement values obtained yields an AUROC of 0.806, which is significantly different from each of the single measurements (Figures 1 & 2).

Even more surprisingly, a simple calibration of the measurement by tgc 43 by the second measurement using tgc 44 (division: v43/v44), again improves the performance. As shown in Figure 5, the corresponding AUROC is 0.813.

The further improvement of calibration algorithms and the inclusion of total serum PSA is still improving the performance. As an example a combination of ANXA3 measurements by tgc 43 and tgc 44 and total PSA is shown in Figure 6. The AUROC of this processing of only ANXA3 and total PSA measurements in serum is 0.840.

Reference List

Duijvesz, D., Luider, T., Bangma, C.H., and Jenster, G., 201 1 . Exosomes as biomarker treasure chests for prostate cancer. Eur. Urol. 59, 823-831 .

Kollermann, J., Schlomm, T., Bang, H., Schwall, G.P., von Eichel-Streiber, C, Simon, R., Schostak, M., Huland, H., Berg, W., Sauter, G., Klocker, H., and Schrattenholz, A., 2008. Expression and prognostic relevance of annexin A3 in prostate cancer. Eur. Urol. 54, 1314-1323.

Schostak, M., Schwall, G.P., Poznanovic, S., Groebe, K., Muller, M.,

Messinger, D., Miller, K., Krause, H., Pelzer, A., Horninger, W., Klocker, H., Hennenlotter, J., Feyerabend, S., Stenzl, A., and Schrattenholz, A., 2009. Annexin A3 in urine: a highly specific noninvasive marker for prostate cancer early detection. J. Urol. 181 , 343-353.

Tavoosidana, G., Ronquist, G., Darmanis, S., Yan, J., Carlsson, L., Wu, D., Conze, T., Ek, P., Semjonow, A., Eltze, E., Larsson, A., Landegren, U.D., and Kamali-Moghaddam, M., 201 1 . Multiple recognition assay reveals

prostasomes as promising plasma biomarkers for prostate cancer. Proc. Natl . Acad. Sci. U. S. A 108, 8809-8814.

Wozny, W., Schroer, K., Schwall, G., Poznanovic, S., Stegmann, W., Dietz, K., Rogatsch, H., Schaefer, G., Huebl, H., Klocker, H., Schrattenholz, A., and Cahill, M.A., 2007. Differential Radioactive Quantification of Protein Abundance Ratios Between Benign and Malignant Prostate Tissues: Cancer Association of Annexin A3. Proteomics. 7, 313-322.

Claims

1 . A method for the in vitro diagnosis of cancer, particularly prostate cancer, comprising at least one measurement of a specific conformation of human Annexin A3 in a body fluid sample, wherein said specific conformation of human Annexin A3 is selected from a first conformation which is present in a first type of vesicles and a second conformation which is present in a second type of vesicles, and wherein the first type of vesicles is different from the second type.

2. The method as claimed in claim 1 , wherein the first and second vesicles are extracellular vesicles.

3. The method as claimed in claim 1 or 2,

wherein the body fluid is selected from the group consisting of urine, exprimate urine, blood, serum, plasma and further blood derivatives, and seminal fluid, preferably from blood serum, plasma and further blood derivatives.

4. The method as claimed in any one of the preceding claims, comprising

(i) a conformation-specific measurement of the first conformation, or

(ii) a conformation-specific measurement of the second conformation, or

(iii) conformation-specific measurements of both the first and the second conformation.

5. The method as claimed in any one of the preceding claims,

wherein the measurement is carried out after chemical and/or physical separation of the first and second vesicles.

6. The method as claimed in any one of the preceding claims, wherein the measurements are carried out in a sample supplemented with metal ions, particularly calcium, and/or an organic solvent, particularly DMSO.

7. The method as claimed in claim 5 or 6, wherein the sample has been subjected to a physical separation procedure, particularly centrifugation.

8. The method as claimed in any one of the preceding claims,

wherein the at least one measurement is carried out by an immunological detection procedure.

9. The method as claimed in any one of the preceding claims, wherein

(i) a conformation-unspecific detection reagent, e.g. a conformation- unspecific antibody is used for the measurement, or

(ii) at least one conformation-specific detection reagent, e.g. a conformation-specific antibody is used for the measurement.

10. The method as claimed in claim 9,

wherein a first and second antibody are used, wherein the first antibody specifically recognizes the first conformation of human Annexin A3 and the second antibody specifically recognizes the second conformation of human Annexin A3.

1 1 . The method as claimed in claim 10,

wherein one of the conformation-specific antibodies is (i) antibody tgc 43 (DSM ACC 2970), (ii) an antibody recognizing the same conformational domain on human Annexin A3 or (iii) an antigen-binding fragment of (i) or (ii).

12. The method as claimed in claim 10 or 1 ,

wherein one of the conformation-specific antibodies is (i) antibody tgc 44 (DSM ACC 2976), (ii) an antibody recognizing the same conformational domain on human Annexin A3 or (iii) an antigen-binding fragment of (i) or (ii).

13. The method as claimed in any one of the preceding claims,

further comprising calibration of the at least one measurement.

14. The method as claimed in claim 13,

wherein calibration of the measurements comprises obtaining mathematical derivatives of the concentration measurements, such as a quotient, product, logarithm etc. and/or using additional other antibodies against human Annexin A3, which recognize further conformations of the protein.

15. The method as claimed in any one of the preceding claims,

wherein the two concentrations of Annexin A3 obtained from the separate measurements are calibrated by at least one additional biophysical or biochemical parameter of the corresponding body fluid.

16. The method as claimed in claim 15,

wherein the additional biophysical or biochemical parameter is selected from the group consisting of density, viscosity, inflammatory markers, ions, in particular calcium ions, phospholipids, cholesterol, sphingolipids and enzymes.

17. The method as claimed in any one of the preceding claims,

wherein the diagnostic performance is improved by combining the two concentrations of Annexin A3 in serum with at least one further cancer marker from other sources of the same patient, such as a tissue sample, in particular a biopsy.

18. The method as claimed in any one of the preceding claims,

wherein the further cancer marker is selected from the group comprising prostate specific antigen (PSA) and variants thereof, PCA3, PCADM-1 and EPCA-2.

19. The method as claimed in any one of the preceding claims, wherein the measurement of Annexin A3 is combined with measuring prostate specific antigen (PSA), free PSA, % free PSA, complexed PSA (cPSA) and/or other variants of PSA.

20. A reagent kit for the measurement of human Annexin A3 in a body fluid sample, comprising a first and a second antibody, wherein the first antibody specifically recognizes a first conformation of human Annexin A3 and the second antibody specifically recognizes a second conformation of human Annexin A3.

21 . A reagent kit for the measurement of human Annexin A3 in a body fluid sample, comprising an antibody specifically recognizing human Annexin A3, and a reagent or reagent combination for separating a first conformation of human Annexin A3 from a second conformation of human Annexin A3 in said body fluid sample.