WO1999023491A1

WO1999023491A1 - Diagnosis of guillain barre syndrome or cancer by detecting integrin alpha6/beta4

Info

Publication number: WO1999023491A1
Application number: PCT/EP1998/006778
Authority: WO
Inventors: Pier Carlo Marchisio
Original assignee: Fondazione Centro San Raffaele Del Monte Tabor
Priority date: 1997-10-31
Filing date: 1998-10-29
Publication date: 1999-05-14
Also published as: IT1295938B1; ITMI972452A1; AU1155799A

Abstract

A method for the determination of α6β4 integrin, of the single subunits or of fragments thereof, in the serum or in other biological of patients Guillain Barré syndrome or neoplasias.

Description

DIAGNOSIS OF GUILLAIN BARRE SYNDROME OR CANCER BY DETECTING INTEGRIN ALPHA6/BETA4

The present invention relates to a method for the diagnosis of Guillain Barre syndrome or neoplasias, based on the determination of integrin ₆β₄, its subunits or fragments thereof from the serum or other biological fluid samples Until today, no diagnostic methods in vitro have been described which are carried out starting from samples of serum or other biological fluids, directed to the determination of ₆β₄ integrin, in pathologies involving "adhesion molecules", from the molecular point of view, i.e. the class of molecules, including integrins, that mediate cell-to-cell or cell-to-extracellular matrix bonds. Integrins are transmembrane heterodimers formed by the non-covalent assembly of an α chain with a β chain. At present, 13 chains and 8 β chains are known, which are combined to form at least 20 heterodimers having different properties [Hynes, 9.0. 1992) "Integrins: versatility, modulation and signaling in cell adhesion". Cell 69:11-25]. U.S. 5,527,679 discloses a method for the determination of β₅ integrin subunit in human biological fluids, said method being used in the diagnosis of neoplasias or pre-neoplasias.

The α₆β₄ integrin (reviewed in Giancotti, (1996) J. Cell Sci. 109:1165-1172) has peculiar aspects, in that the β₄ chain, contrary to all the other β chains, has a large-sized intra-cytoplasmatic portion consisting of more than 1 ,000 amino acids. Notably this cytoplasmatic portion is characterized by two tandemly repeated fibronectin type III domains separated by a bridge sequence which comes in four splicing variants. The α₆β₄ integrin, which is the major receptor for multiple isoforms of laminin in normal and transformed epithelial cells, is the main adhesion component of hemidesmosomes

It is known from the literature that the regulation of ₆β₄ integrin expression is altered in different types of tumors. For example, most carcinomas characterized by invasive and metastatic growth [Kimmel, K.A. and T.E. Carey, (1986) Cancer Res. 46:3614-3623; Savoia, P., L. Trusolino, E. Pepino, O. Cremona and P.C. Marchisio (1993) J. Invest. Dermatol. 101 :352-358.], as well as neoplastic and tumorigenic epithelial cells grown in vitro, show a pericellular expression of α₆β₄ with loss of the characteristic polarization toward the basement membrane. Most epithelial tumors expose α₆β₄ at the surface of the component cells [Rabinovitz, I. and A.M. Mercurio (1996) Biochem Cell Biol. 74:811-821]. Thyroid tumors also, which are predominantly β₄-negative, turn β₄-positive only in a highly malignant subpopulation [Serini, G., L. Trusolino, E. Saggiorato, A. Angeli,

F. Orlandi and P.C. Marchisio. (1996) J. Natl. Cancer. Inst. 88:442-449]. US

5 320 942 discloses a method of diagnosing abnormal epithelial tissue in vitro which is based on immunodetection of α₆β₄ on tissue preparations. The expression of α₆β₄ is commonly accepted as a mechanism highly favourable to invasiveness due to the polyvalent nature of α₆β₄ as a receptor for multiple laminin forms. This favours the invasive and metastatic growth through barriers of basement membranes containing different forms of laminin, such as those forming vascular and lymphatic subendothelia. This phenomenon is particularly noted in a subpopulation of highly malignant thyroid carcinomas where β₄ neoexpression is related with a highly unfavourable prognosis and can easily be observed even in simple fine needle-aspiration biopsies (Serini et al., 1996). On the other hand, α₆β₄ also plays an important role in Guillain Barre syndrome, an acute peripheral neuropathy which is the most widespread cause of generalised paralysis. The disease results from aberrant immune responses directed against components of peripheral myelin, in particular against Schwann cells. However, the primary damage causing the disease . has not yet been clarified; hence, reliable serum markers are not yet available.

The α₆β₄ integrin was identified as a primary receptor for laminin expressed by Schwann cells only when these are in contact with single nerve fibers (Niessen et al., 1994). Therefore α₆β₄ is important for the correct assembly of the myelin sheath.

Until today, early diagnosis of Guillain Barre syndrome has been based on clinical experience, as reliable markers to validate such diagnosis are not yet available. On the basis of the novel role of α₆β₄ it has now been found that the individual α₆ and β₄ subunits or the fragments thereof, circulate in the early phases of the disease described above, and that such circulation can be related with the subsequent pathological development. Moreover, it has been found that in Guillain Barre syndrome, the determination of integrin allows to check the evolution of the disease as well as the level of the myelin damage.

In a set of independent assays it has also been found that in many tumors the presence of α₆β₄ in the bloodstream may reveal the onset of the metastatic process and can provide information on the presence of circulating metastatogenic cells even before the primary tumor from which they originate is clinically detectable.

The invention therefore provides a method for the diagnosis of Guillain Barre syndrome or neoplasias in which integrin α₆β₄, single α₆ and β₄ subunits or fragments thereof, are determined from the serum or any other biological fluid, isolated from the body of patients, according to the following steps: a) contacting the serum or other biological fluid with a first monoclonal or polyclonai antibody directed against an α₆β₄ antigenic determinant, so as to form a complex; b) contacting the complex of step (a) with a second antibody, either monoclonal or polyclonai, which can be the same or different from the first, which is capable of binding the complex and to make the complex detectable, either directly or by coupling with one or more detectable molecules; c) quali-quantitatively determining the formed complex.

The first antibody can be a monoclonal directed against α₆β₄ integrin, the single α₆ and β₄ subunits or peptide fragments thereof, or a polyclonai against the same antigens, possibly reconstituted starting from a monoclonal mixture. The use of polyclonai antibodies recognising as wide a spectrum of peptide fragments as possible is preferred.

The second antibody can, for example, be coupled with a radioactive isotope, an enzyme, a fluorogen, a chemiluminescent or a luminescent, enabling also an automated detection of the results.

The use of a biotinylated antibody, coupled with a streptavidin-derivatized enzyme, is preferred.

In a preferred embodiment of invention, a solid phase immunologic assay based on double-determinant ELISA is used ("Sandwich ELISA").

According to this embodiment, the first catcher antibody is represented by a polyclonai which is reconstituted from monoclonals against peptide fragments derived from the lysis of α₆ and β₄; the second antibody, on the other hand, consists of the same monoclonals, which have been biotin- conjugated.

The reaction between streptavidin-conjugated peroxidase and o- phenylenediamine can be used for the detection of the complex.

Therefore, the invention also relates to the use of antibodies raised to α₆β₄, the single α₆ and β₄ subunits or fragments thereof, for the determination of α₆β₄ of the single α₆ and β₄ subunits or fragments thereof, in the serum of patients with Guillain Barre syndrome (hereinafter "GBS patients") or with neoplasias.

A further object of the invention is provided by a diagnostic kit for the determination of α₆β₄, of the single α₆ and β₄, subunits, or of fragments thereof, containing the first and the second antibodies of claims 1-2 and the detection reagents in suitable containers, together with carriers, excipients and stabilizers.

The method of the invention can also be applied to the in vitro detection of circulating metastatogenic cells or fragments thereof starting from blood samples; therefore, it is highly useful in the prevention and early diagnosis of epithelial tumors, which can be diagnosed well before than with conventional methods.

The results of the tests carried out according to the method of the invention, the details of which are described hereinafter in the examples, are reported in the figures.

Figure 1 shows that all the α₆- and β₄-positive sera belong to GBS patients and none to the control groups.

These results proved that immunoreactive α₆ and β₄ were found in about 66% of GBS sera analyzed and that virtual matching occurred between α₆ and β₄ and this suggested that release in the bloodstream interests both components of the heterodimer α₆β₄.

Figure 2 shows the analysis of the profile of β₄ serum levels in eight GBS patients. In three of the latter there was a preceding flu-like illness, in two there was only leg weakness, in one legs were significantly weaker than arms and in two patients there was flaccid paralysis of all limbs.

The data of Figure 2 shows that β₄ release occurs for a variable length of time during disease progression. These results clarified how some of the measurements carried out at different times during the disease progression, as reported in fig. 1 , could be false-negative.

In fact, to obtain an optimum control of the early progression of the disease, a constant monitoring of the β₄ serum levels is necessary, from the onset of the first symptoms. In other words, by optimizing the monitoring of α₆β₄ in the patients under observation, it is possibile to obtain a correlation between α₆β₄ (respective subunits or fragments thereof) levels and progression of the disease, better than the correlation reported in fig. 1.

The detection of ₆β₄ according to the method of the invention was applied to different tumors, giving the results reported in the following Table.

Table

Tumor n. n. positive n. negative % positive

Head/neck 71 18 53 25

Breast 97 30 67 31

Digest, tr. 63 16 47 25

Lung 36 10 26 28

Ovary 20 10 10 50

Urinary bladder 3 1 2 33

Kidney 4 2 2 50

Uterus 3 1 2 33

Prostate 12 5 7 42

Melanoma 24 8 16 33

Ewing sarcoma 5 0 5 0

The mean of positive carcinomas is nearly 30% on the total, with values of

50% in ovary (shown in fig. 3) and kidney tumors. Melanomas, which are not usually β₄ positive from the histochemical point of view, diffuse β₄ in the serum in about one third of the cases.

Also in the case of tumors, to obtain an optimum result, a constant monitoring of α₆β₄ concentration is necessary to obtain a valid reference pattern and a prognosis of the disease progression, in particular of the formation of metastases from circulating cancer cells.

The following examples illustrate the invention in greater detail.

Example 1 -

ELISA ("Enzyme-Linked Immunosorbent Assay") test procedure

- Antibodies -

A reconstituted polyclonai antibody to β₄ (pβ₄) was used, prepared by mixing the following four mouse monoclonals:

- AA3 [Kajiji, S., Tamura, R.N., and Quaranta, V. (1989) EMBO J. 8, 673-

680];

- 3E1 [Holmes, E. and Engvall, E. (1993) Anal. Biochem. 214, 100-105]; - 346-11 A [Kennel, S.J., Foote, L.J. and Lankford, P.K. (1981 ) Cancer Res.

41 , 3465-3470];

- 439-9B [Falcioni, R., Sacchi, A., Resau, J. and Kennel, S.J. (1988) Cancer

Res. 48, 816-821], and by two monoclonal to ₆

- (pα₆) GOH3 [Sonnenberg, A., Modderman, F.W. and Hogervorst, F. (1986) Nature 336:487-489];

- MAR6 [Bottini, C, Miotti, S., Fiorucci, S., Facheris, P., Menard, S. and Colnaghi M.I. (1993) Int. J. Cancer 54, 261-267]. In addition to the monoclonal antibodies listed in Table 1 , the rabbit serum

SP90395, raised to the 17 C-terminal amino acids of the β₄ cytoplasmatic chain, was also used.

- Biotinylation of the antibodies -

Each purified antibody was diluted with H2O to a concentration of 2 mg/ml and dialyzed against H2O to remove PBS (phosphate-buffered saline). After dialysis, the final antibody concentration was about 1.5 mg/ml. 1 ml of the dialysed antibody solution was added with 110 μl of 1 M Na2CO3 (pH 8.8) and with 110 μl of 6-aminocapryloyl-N-hydroxysuccinimidic ester (AH-BNHS, 1 mg/ml in DMSO, dimethylsulfoxide), in succession. This solution was incubated for 4 hours at room temperature. After incubation, 125 μl of 1 M lysine were added and the solution was incubated for a further 3 hours at room temperature, dialysed against PBS (pH 7,4), aliquoted and stored at - 20°C.

- Procedure - 96-Well plates (NUNC) were coated with 10 μg/ml of the catcher antibody by incubation for 12 hours at 4°C. In each well, previously washed with PBS- 1 % BSA, the residual sites were blocked with PBS-3% BSA for 2 hours at room temperature. After rinsing three times with PBS-1 % BSA-0.1 % Tween 20 (assay buffer) the wells were added with 100 μl of standard antigene at increasing dilutions (FG. 2 pancreatic carcinoma cell lysate; positive control containing both α₆ and β₄) or with 100 μl of patient serum at increasing dilutions (1 :2 to 1 :16) in assay buffer containing 50% FBS (fetal bovine serum). Incubation was standardized in 2 hours at room temperature. Subsequently, after repeated (8x) and thorough rinsing in assay buffer,

100 μl of 1 , 000-fold diluted biotin-conjugated tracer mAb were added to each well and the plate was incubated for 1 h at room temperature. After further rinsing 8 times in assay buffer, each well was added with an aliquot of 100 μl of 500-fold diluted streptavidin-conjugated peroxidase (Sigma), followed by incubation for one hour at room temperature. After rinsing 3 times in assay buffer and 5 times in PBS, 100 μl of o-phenylenediamine was added to the wells and the plate incubated for 30 min at room temperature.

The enzyme reaction was stopped with 100 μl of H2SO4 and the absorbance at 492 nm was measured in a microplate reader equipped with a 490 nm interference filter. Serum antigens levels were determined as absorbance at 492 nm at 1 :1 (v:v; serunτassay buffer). All experiments were performed in duplicate. The cut-off value above which absorbance levels were considered positive was determined as the mean + 2SD of levels from control sera.

Example 2 -

Application of the method on GBS patients.

The diagnosis of acute GBS was made according to the current diagnostic criteria. Serum samples were obtained from 61 GBS patients from a consecutive series of GBS patients admitted at the Dept. of Neurology, San Raffaele Hospital, from 1991 to 1996. Serum samples were drawn at various time intervals after the onset of motor symptoms as indicated in parentheses: 36 patients (2-7 d), 19 patients (8-14 d), 6 patients (15-21 d). Five patients, admitted at the beginning of 1996, underwent blood sampling every 2-3 d to study the profile of α₆β₄ release.

Sixty patients with non-GBS neuropathies, namely 18 diabetic, 18 vasculitic, 5 toxic, 10 subacute paraneoplastic neuropathy, 9 Charcot-Marie-Tooth type 1 (CMT-1 ) and 40 healthy blood donors served as controls. In the non-GBS neuropathies 9 sera of CMT-1 were included, as a group of chronic demyelinating non-inflammatory neuropathies.

In all the tested sera, acute phase reactants, such as erythrocyte sedimentation rate, mucoproteins, complement components and C-reactive protein, were within the normal limits.

All the serum samples were rigourously blind tested.

The person performing the test and the one analysing the results were not aware of the serum source during data acquisition. Samples themselves were a random mixture of sera from normal individuals and from GBS patients.

Some preventive tests were carried out using a single catcher monoclonal antibody and the same antibody as biotinylated tracer. Other tests were carried out using different antibodies as catcher or tracer. The best results were obtained using a polyclonai consisting of a mixture of four different monoclonals (pβ₄) used both as catcher and tracer. The whole lot of 61 sera was assayed with pβ₄. The same sera were used for the parallel determination of α₆ using the combination GOH3 and MAR6. All the ₆ and β₄ positive sera (Fig. 1 ) belonged to patients which had independently been diagnosed as affected by Guillain Barre syndrome; none of the positive sera belonged to the control group. The sera of the 9 control Charcot-Marie-Tooth type 1 patients fell within the values of the control groups. Example 3 - Application of the method on patients with carcinoma -

Only β₄ was searched in this study.

The method described above was used to test about 500 sera of cancer patients which had been subjected to random blood sampling. Some of these patients had already underwent surgery and/or chemo-radiotherapy. Mammary, lung, intestinal, hepatic, ovary, head and neck, prostate, uterus and ovary carcinomas were tested and the obtained results are reported in Table.

Claims

1. A method for the diagnosis of Guillain Barre syndrome or neoplasias in which integrin ╬▒₆╬▓₄, the single ╬▒₆ and ╬▓₄ subunits or fragments thereof, are determined from the serum or any other biological fluid isolated from the body of patients, according to the following steps: a) contacting the serum or other biological fluid with a first monoclonal or polyclonai antibody directed against an ╬▒₆╬▓₄ antigenic determinant, so as to form a complex; b) contacting the complex of step (a) with a second antibody, either monoclonal or polyclonai, which can be the same or different from the first and which is capable of binding the complex and to make the complex detectable, either directly or by coupling with one or more detectable molecules; c) quali-quantitatively determining the formed complex.

2. A method as claimed in claim 1 , in which:

- the first antibody is a polyclonai reconstituted from a mixture of monoclonals directed against peptide fragments from ₆╬▓₄ lysis;

- the second antibody is the same polyclonai in which each monoclonal had been biotin-conjugated;

- the formed complex is revealed by reaction with streptavidin-co╧Çjugated peroxidase.

3. A method according to claims 1-2, for the in vitro determination of circulating metastatogenic cells from blood samples.

4. A diagnostic kit for the determination of serum ╬▒₆╬▓₄, single ₆ and ╬▓₄ subunits or fragments thereof, containing the first and second antibodies of claims 1-2 and the detection reagents in suitable containers, together with carriers, excipients, stabilizers.

5. The use of antibodies directed against ╬▒₆╬▓₄, single ╬▒₆ and ╬▓₄ subunits or fragments thereof, for the determination of ╬▒₆╬▓₄, single ╬▒₆ and ╬▓₄ subunits or fragments thereof, in the serum of patients affected by Guillain Barre syndrome or by neoplasias.