BG65682B1 - Conjugates of antibodies and dyes for structural outlining the angiogenesis in intraoperative depiction of tumor dimensons - Google Patents

Abstract

Conjugates are described of antibodies and dyes that are suitable for binding to the structures of newly formed blood vessels, as well as the use of such conjugates for intraoperative depiction of pathological angiogenesis.

Description

(54) HA ANTIBODES AND DYES CONJUGATES FOR THE STRUCTURAL DISTRIBUTION OF ANGIOGENESIS IN THE INTRO-OPERATIVE DISPOSAL OF TUMOR DIMENSIONS

Technical field

The present invention relates to conjugates of antibodies and dyes that are suitable for binding to the structure of newly formed blood vessels and the use of these antibodies in the intraoperative imaging of pathological angiogenesis.

BACKGROUND OF THE INVENTION

In adult organisms with very few exceptions (for example, the cycle of women of childbearing age) do not form new blood vessels. The process of formation of new blood vessels is characterized as angiogenesis and manifests itself in response to certain signals.

Angiogenesis is a process that occurs in the border areas of the outbreaks of disease. From the center of the disease outbreak are released factors that are defined as the border area of the outbreak.

These factors are also known to stimulate angiogenesis.

When these angiogenesis promoters reach healthy tissue in the border areas of the outbreak, then the blood vessels not included in the outbreak are stimulated to form vascular embryos of new blood vessels. The blood vessels formed by these vascular embryos form a new circulatory system in the border area of the outbreak. Through this process, an adequate supply of the disease outbreak is always ensured. Of particular importance, tumor growth and their metastases have been found to be dependent on their ability to induce angiogenesis.

Surgical therapy is now a standard form of treatment for localized foci of disease. It is of great importance in the successful treatment of tumors. However, it has been found that, despite improved surgical techniques, local recurrences occur because the anatomical features in the human body rarely allow the outbreak of the disease to be eliminated to the maximum extent. In many organs (such as in the brain), the removal should be maximized in order to maintain healthy tissue. The risk of damage to healthy organs increases with the radical nature of surgery.

However, histological examinations of the border areas of the tumor after subsequent surgical removal show that many of the tumors cannot be completely removed and parts of the tumor remain in the body. Further development of the tumor or its metastases results from these tumor parts. One method to accurately determine the boundary between the outbreak of disease and healthy tissue during surgery would allow complete removal of the disease site and further normal development of healthy tissue.

Dyes for imaging outbreaks of disease have long been known (Poon WS et al., J. Neurosurgery (1992) 76: 679-686; Haglund MM et al., Neurosurgery (1996) 38: 308 - 317). These will preferably absorb directly from the tumor cells or saturate non-specific outer cell space around the tumor. As the mechanism of saturation can also be demonstrated in healthy tissue, the specificity and sensitivity of the substances used is limited.

To date, there are no known compounds that can be used to delineate the outbreak of disease by selectively depicting the outbreak boundary.

Angiogenesis is preferably carried out in the border region of the outbreak. By displaying angiogenesis, the border with healthy tissue can be depicted. Antibodies for proving angiogenesis at the outbreak of the disease have long been known and can be used to display newly formed blood vessels in histologic tissue sections, to demonstrate various proteins at the outbreak of the disease, or as carrier molecules for therapeutic substances.

However, antibodies in the combination are unknown

65682 Dye dye, the so-called antibody and dye compounds, which can be used to delineate the outbreak of disease by selectively depicting the outermost regions of the disease outbreak.

It is an object of the present invention to provide conjugates of antibodies and dyes for intraoperative imaging of tumor sizes. The antibodies of the antibody and dye compounds of the invention are intended for structures that are specific to the process of angiogenesis. The antibodies and dyes conjugates of the invention encompass dyes which, by absorption (in a particular tissue area), make them optically imaging.

As angiogenesis is most pronounced in the borderline areas of the outbreak, they receive the strongest signal. The antibodies associated with the dyes according to the invention are suitable for imaging the border, the outline of the outbreak of the disease, the so-called border region, with healthy tissue by intraoperative, optical diagnosis. In this way it is possible to completely eliminate the outbreak of the disease from the healthy tissue and to further protect it.

Antibodies are known to target molecules that are highly expressed in angiogenetically active tissue and are restricted to a very limited level in restricted tissue (WO 1996/001653).

Of particular interest in antibody and dye conjugates are antibodies that target receptors for vascular growth factors, endothelial cell receptors that bind to inflammatory beds, and proteins that are specifically expressed in the formation of new blood vessels (Brekken et al., Cancer Res. (1998) 58: 1952-9 and Schold SC Jr et al., Invest. Radiol. (1993) 28: 488-96).

Antibodies or portions of antibodies that are intended for the EDB-fibronecin protein are preferred. EDB-Fibronectin (EDBFN), also known as oncofetal fibronectin, is one variant of fibronectin that is formed specifically around newly formed blood vessels in the process of angiogenesis. The particular advantage of antibodies against

EDB-fibronectin consists in the fact that intra-operative injury at the outbreak of the disease does not lead to any new formation of EDB-fibroprotectin in healthy tissue. Thus, they retain their specificity during surgery. Antibodies to growth factor receptors or to mediators (mediators) of inflammation in endothelial cells, which are also specifically expressed in the border regions of the tumor, can be re-formed in healthy tissue in the immediate vicinity of the outbreak during surgery.

Particularly preferred conjugates of antibodies and dyes of the invention are antibodies L 19 and E 8 against EDB-fibronecin (Viti et al., Cancer Res (1999) 59: 347-352).

Such antibody and dye conjugates are also an object of the present invention.

The known antibodies bind to dyes, the concentration of which in the tissue can be detected optically and make it possible to differentiate intraoperatively the boundary regions of the outbreak.

The advantage of the conjugates of antibodies and dyes according to the invention, therefore, is that they can be used for selective fluorescence staining of tissues in the neoangiogenetic stage. Fluorescence staining is tumor specific and produces a fluorescent signal that can be detected in a high signal to background ratio.

Antibody and dye conjugates are also known to produce a fluorescent image for the purposes of percutaneous, non-invasive tumor imaging (Neri D et al., Nature Biotechnology (1997) 15: 1271-1275).

However, conjugates of antibodies and dyes are known, which concentrate predominantly at the border regions of the outbreak.

Protein and dye conjugates are also known for intraoperative tumor imaging.

The disadvantage of these conjugates is that the tumor cells provided in particular, both hypoxidally and metabolically, absorb the compounds. Because the tissue at the border regions of the tumor is well vascularized and therefore the cells are oxygenated and nutrient-dense, it becomes impossible

65682 B1 saturation with known protein and dye compounds.

SUMMARY OF THE INVENTION

The antibody and dye conjugates of the invention are independent of the metabolic state of the outbreak.

To the extent that the optical detection of the boundaries of a disease outbreak can be accomplished in various ways, it is preferable to fully record the induction by appropriate excitation light, fluorescent radiation, of a specific dye. Typically, depending on the emission wavelength, fluorescence can be detected visually directly macroscopically or microscopically and optionally displayed digitally by a picture-based detector system on screen.

Visually, fluorescence radiation can be detected in the spectral range from 400 to 650 nm. One wavelength of 450 to 600 nm is particularly preferred. The advantage of using light in the visible region is that fluorescence detection is possible with the help of simple technical means. The excitation light, which is obtained with a suitable laser or laser diodes, is introduced into the light line and through it into the diagnosed area. Intraoperative tumor size determination is performed by irradiation of a wide area of the site. A filter (eg filter glasses used by the investigator) blocks the reflected excitation light and only the specific dye fluorescence is observed (macroscopic observation). Alternatively, fluorescence can be recorded by operating microscope (microscopic observation). By limited penetration of VIS light into the depth of the tissue (several millimeters), superficial localized neoplasms of blood vessels can be covered.

Another advantage of the spectral region of visible light lies in the limited penetration of depth into the tissue and the emission from the tissue. In this way, the detected signal is not compromised by a signal received from the deeper parts of the tissue and can immediately become tissue.

It is also an object of the present invention to provide conjugates of antibodies and dyes whose dyes induce an optical signal in the visible light region.

The use of antibody and dye conjugates in which dyes are absorbed in the spectral region close to infrared light (NIR; 600-900 nm) makes it possible to locate newly formed blood vessels in the deeper layers of tissue (up to 1 cm), the NIR light being absorbed less than the tissue and thus having a greater depth of penetration into the tissue. Visual observation of fluorescence is not possible and can be accomplished with the use of a CCD (Carge Coupled Device - Camera), which is placed at a specific location of the tissue under study. Both macroscopic and microscopic modes are possible. The advantage of using dyes in antibody and dye compounds that are absorbed and fluoresced in the NIR-spectral region is when it is necessary to conduct studies in closed areas (eg through blood).

From a photophysical point of view, antibodies and dyes conjugates are suitable for dyes having an absorption peak in the spectral range of 400 to 800 nm and at least one fluorescence peak in the range of 500 to 900 nm.

It is also an object of the present invention that antibodies and dyes conjugates are characterized in that the dye first induces a fluorescent signal when using a particular wavelength region in the visible region or near infrared light.

Antibody and dye conjugates include dyes with visually detectable fluorescence, which may be, for example, of the following classes:

Fluorescein, fluorescein - isothiocyanate, carboxyfluorescein or calcein, tetrabromfluorescein or eosin, tetraodofluorescein or erythrosine, difluorofluorescein, such as Oregon Green ™ 488, Oregon Green ™ 5004 or Oregon Green ™ 5004 or Oregon Green ™ 514 US 5, 442, 045), carboxyrodamine dyes (e.g. Rhodamine Green ™ Dyes) (US

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5, 366, 860), 4,4-difluoro-4-boron-3a, 4adiazazines, such as Bodipy FL, Bodipy 493/503 or Bodipy 530/550 and derivatives thereof (US 4,774, 339, US 5,187,288, US 5,248,782 U.S. Pat. No. 5,433,896, U.S. Pat. No. 5,451,663), cyanine dyes, such as 7-amino-4-methylcomarine, DTPA or tetraaza-macrocyclic (cyclic, helical) metal complexes with terbium or europium, or tetrapyrrole dyes, in particular porphyrins.

Antibody and dye conjugates involving infrared dyes, for example, are those of the following classes:

polymethine dyes, such as dicarboxycyanin, tricarboxycyanin, merocyanine and oxonol dyes (W0 1996/017628), rhodamine dyes, phenoxazine dyes or phenothiazine dyes, tetrapyrrole dyes, in particular benzoporphyrins, in particular benzoporphyrins.

Preferred near-infrared dyes in the conjugates of antibodies and dyes are cyanine dyes with an absorption maximum of between 700 and 800 shi, in particular indodes and indotricarbocyanins.

Mainly preferred in the conjugates of antibodies and dyes are the dyes of the above classes having one or more carboxyl groups which, upon chemical activation, bind to the amino groups of the antibodies or antibody fragments. Also preferred are those derivatives which contain maleimido or bromoalkyl radicals so that covalent coupling with the sulfide groups of the amino acid cysteine follows.

Dyes containing isothiocyanate groups which also react with amino groups are also preferred.

Therefore, the dyes of the conjugates of antibodies and dyes should have high photostability and, when irradiated with light, do not discolor (photobleaching) to provide a constant signal throughout the study.

In this connection, it is an object of the present invention to have conjugates of antibodies and dyes, which concentrate predominantly in the border region of the cellular tissue of the disease outbreak and make it possible to optically display its border regions, i.e. its size.

It is an object of the present invention to specifically conjugate antibodies and dyes of general formula I

B - (F) _n (I) in which

B stands for an antibody or antibody fragment with a strong link to ED - BFN,

F F is one mosquito repellent dye, fluorescein, carboxyfluorescein, or difluorofluorescein, tetrabromfluorescein, tetraodifluorescein, rhodamine, carboxyrodamine, carboxyrodol, 4,4-difluorozole, 4,4-difetro, of terbium or europium with DTPA or cyclene and their derivatives and

η stands for 1 to 5.

Particularly preferred and therefore object of the present invention are also antibodies and dyes conjugates whose dye is one cyanine dye, one merocyanine dye, one oxonol dye, one styryl dye or one squirrel dye.

Particularly preferred and therefore object of the present invention are also antibodies and dyes conjugates in which the dye moiety is a cyanine dye, in particular a carbocyanine, dicarbocyanine or tricarbocyanine.

The invention relates in particular to conjugates of antibodies to dyes in which a dye - (F) _n of general formula (I) means a cyanine dye of general formula (II)

wherein

D represents a radical of formulas III or IV

bonding to radical B, and

65682 Bl

B means groups V, VI, VII, VIII or IX

V!

VIII

IX in which

R ¹ and R ² denote sulfoalkyl of 1 to 4 carbon atoms, a saturated or unsaturated, branched or unbranched alkyl chain of 1 to 50 carbon atoms which may optionally be substituted by 1 to 15 oxygen atoms, and / or up to 3 carbonyl groups and / or with up to 5 hydroxy groups,

R ³ means the group -COOE ¹ , -CONE'E ² , NHCOE ¹ , -NHCONHE ¹ , -ΝΕΈ ² , -OE ¹ , -OSO ₃ E ', SO ₃ E', -SO ₂ NHE 'or -E ¹ , as

E ¹ and E ² independently represent a hydrogen atom, a sulfoalkyl of 1 to 4 carbon atoms, saturated or unsaturated, branched or unbranched alkyl of 1 to 50 carbon atoms, which may optionally be substituted by up to 15 oxygen atoms, and / or be interrupted by up to 3 carbonyl groups, and / or replaced by up to 5 hydroxyl groups,

R ⁴ represents a hydrogen atom or a fluorine, chlorine, bromine or iodine atom, b means 2 or 3,

X represents an oxygen atom, sulfur or group = C (CH ₃ ) ₂ or - (CH = CH) -, and

L means a direct bond or linker which represents a single or branched chain of up to 20 carbon atoms which may be substituted by one or more -OH, -COOH, SOj groups, and / or optionally may be single or repeatedly interrupted by one -O-, -S-, -CO-, -CS-, -CONH-, NHCO-, -NHCSNH-, -SO ₂ , PO ₄ - or one -NH group, or with an aryl ring.

The antibody and dye conjugates according to the invention can be used both alone and in the form of preparations as medicaments.

When using conjugates of antibodies and dyes as medicaments, they must be in the form of pharmaceutical preparations which, in addition to the antibody-dye compound, enter into enteral or parenteral administration with appropriate pharmaceutical, organic or inorganic inert materials, such as water, gelatin, arabic gum, milk sugar, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, etc. Pharmaceutical preparations may be in solid form, such as tablets, dragees, suppositories , Capsules or in liquid form such as solutions, suspensions or emulsions. Optionally, they also contain adjuvants such as preservatives, stabilizers, wetting agents or emulsifiers, salts for changing the osmotic pressure or buffers.

For parenteral administration, injectable solutions or suspensions, in particular aqueous solutions of antibody and dye conjugates, are suitable.

As carrier systems, auxiliary agents active at the boundary surface, such as gallic acid salts or animal or plant phospholipids, but also mixtures thereof as well as liposomes or their constituents may be used. Suitable for oral administration are tablets, dragees or capsules with talc and / or hydrocarbon carriers or hydrocarbon binders such as lactose, corn or potato starch. Liquid forms, such as syrups, which may optionally contain a sweetener may also be used.

The dosage of antibody and dye conjugates may vary according to the patient's prescription, age and weight, the type and severity of the disease to be treated, and other similar factors. The dosages of dye antibody compounds used to determine boundary regions, i. the sizes are 0.5-1000 mg, preferably 50-200

65682 Bl mg, at which time the dose can be taken once as a sufficient single dose or divided into 2 or more daily doses.

The above-described administration and administration forms are also contemplated by the present invention.

Therefore, the invention also relates to pharmaceutical agents which include one or more conjugates of antibodies and dyes, intended for intraoperative imaging of the border regions of a disease outbreak, wherein the pharmaceutical agents can be used alone or in admixture with suitable solvents, buffers and / or carriers.

The antibodies and dyes conjugates of the invention are useful in the surgical intervention of diseases related to angiogenesis, such as malignant tumors and their metastases, primary tumors, precancerous tissue changes, endometriosis, hemangiomas, ectopic pregnancy.

It is also an object of the present invention to use conjugates of antibodies and dyes and agents for the intraoperative imaging of disease outbreaks, preferably for micro- and macroscopic, intraoperative imaging of the border regions of a disease outbreak, and the use of antibody conjugates and dyes for the preparation of agents for the surgical treatment of angiogenesis-related diseases such as malignant tumors and their metastases, precancerous tissue changes, endometriosis, hemangiomas and extra eye pregnancy.

Examples of carrying out the invention

Getting dyes

The dyes are prepared by methods known in the literature. Suitable dyes for the preparation of antibody and dye compounds are those which contain carboxyl groups or isothiocyanate groups capable of binding to the amino groups of the antibody. Cyanine dyes, for example, are preferred (Mujumdar SR et al. (1996) 7: 356-362; Flanagan JH et al. (1997) 8: 751-756 and Licha K. et al. (1996) Proc. SPIE Vol. 2927, 192-198).

Carboxyl group dyes are activated by conversion to a reactive ester (e.g., N-hydroxysuccinimidester) by known methods. Dyes with isocyanate groups can be used directly. The reactive derivatives are reacted with antibodies by adding to a buffer solution or mixtures of organic solvents (eg dimethylformamide (DMF)) or dimethyl sulfoxide (DMSO) and buffer solution. In this case a 3- to 100-fold molar excess of the dye is used. The unreacted moiety is separated after completion of the reaction by ultrafiltration and / or chromatography.

The following dyes are obtained in a similar manner.

Preparation Example 1

Bis-1,1 '- (4-sulfobutyl) indocarbocyanine-5-carboxylic acid-N-hydroxysuccinimide ester

The preparation of bis-1,1 '- (4-sulfobutyl) indocarbocyanine-5-carboxylic acid proceeds from 1- (4-sulfobutyl) -2,3,3-trimethyl-3H-indolinine and 1- (4-sulfobutyl) -

2,3,3-Trimethyl-5-carboxy-3H-indolenine (Cytometry 10, 11-19, 1989, Taianta 39, 505-510, 1992) based on methods known in the literature. For the conversion to the N-hydroxysuccinimidester, 0.1 mmol of dye (67 mg in 10 ml of DMF) is usually mixed with 0.5 mmol of Nhydroxysuccinimide and dicyclohexylcarbodiimide (DCC) and stirred for 24 hours at room temperature. After the addition of 50 ml of ether, the precipitate was removed by filtration, dissolved twice with a small amount of DDF and precipitated with ether, finally dried in vacuo (89% yield).

Preparation of antibody-dye conjugate

Preparation of the bis-1,1 '- (4-sulfobutyl) indocarbocyanin conjugate with L19 antibody

The L19 antibody (1 mg in 1 ml of sodium acetate buffer 50 mM, pH 8.2) was mixed with N-hydroxysuccinimidester (75 micromol solution at 4 mg / ml in DMSO) and stirred for 2 h at room temperature. Purification was performed by gel filtration through a PD 10-cartridge (pharmaceutical) and concentration with Centricon-10 tubes (Amicon) while maintaining the solution with about 1 mg / ml antibody.

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Absorption maximum: 555 nm

Fluorescence maximum: 582 nm.

The following example clarifies the bioavailability of the antibody and dye compounds of the invention without limiting the use cases.

Example of Annex 1

Fluorescence imaging of tumor-bearing nude mice in vivo and microscopic examination of tumor tissue ex vivo 10

The picture-represented properties of the compounds of the invention are examined after injection into tumor-bearing nude mice. 0.1 micromol / kg to 2 micromol / kg of the substance is injected intravenously and its concentration in the tumor area is monitored for a period of 0 to 48 hours. The fluorescence of the substance is induced by irradiating the animals with light having a corresponding wavelength, which is obtained by a monochrome 2 0, directly with a laser (diode laser, solid-state laser) or by a filter with a polychromatic emission from a mercury or helium lamp. When preparing compounds of Example 1, light from an Nd: YAG 25 laser with a wavelength of 540 nm is used to excite the full and comprehensive illumination of the experimental animals and detect fluorescence at a wavelength of> 580 nm using one an intensive CCD camera at occupation of total fluorescence obtained from all parts of the body. Fluorescence is read in parallel visually and photographically. From the tumor material, microscopically prepared preparations are prepared (Zeiss Axiovert 35 Mikroskop mit Cu3 - Filtersatz).

After injection of 1 micromol / kg of the indicated 1 antibody and dye compounds in P9-teratocarcinoma nude mice, it is possible to detect an increased fluorescence signal after 4 hours compared to normal tissue recorded by total body phosphorescence. .

After preparation of the skin preparation and the superficial layers of the tumor, the fluorescence of the border regions of the tumor is calculated. Microscopic evaluation of the tumor layers shows an increased fluorescence that corresponds to blood vessels in the tumor border regions.

Claims (10)

Claims 1. Antibody and dye conjugates, characterized in that they are concentrated predominantly in the border regions of the cellular tissue of a disease outbreak, thus making it possible to optically display the dimensions of the outbreak of the disease, characterized in that the dye is compound of general formula (I) B - (F) _n (I) in which B stands for an antibody or antibody fragment with good binding to EDB - fibronectin, F means a cyanine dye of general formula (II) wherein D stands for one radical (III) or (IV) stands for the point of attachment to radical B, and B stands for V, VI, VII, VIII or IX 65682 Bl in which R ¹ and R ² denote sulfoalkyl of 1 to 4 carbon atoms, one saturated or unsaturated, branched or straight alkyl chain of 1 to 50 carbon atoms, which may optionally be substituted by up to 15 oxygen atoms, and / or by up to 3 carbonyl groups and / or with up to 5 hydroxyl groups, R ³ means -COOE ¹ , -CONE'E ² , -NHCOE ¹ , -NHCONHE ¹ , -NE'E ² , -OE ¹ , -OSO ₃ E ', -SO ₃ E', SOjNHE ¹ or -E ¹ , as E ¹ and E ² independently of one another denote a hydrogen atom, a sulfoalkyl of 1 to 4 carbon atoms, saturated or unsaturated, branched or unbranched alkyl of 1 to 50 carbon atoms, which is optionally interrupted by up to 15 oxygen atoms, and / or with up to 3 carbonyl groups, and / or may be substituted with up to 5 hydroxyl groups, R ⁴ represents one hydrogen atom or one fluorine, chlorine, bromine or iodine atom, b means 2 or 3, X and Y denote an oxygen atom, sulfur or group = C (CH ₃ ) ₂ or - (CH = CH) -, and L means one direct bond or one linker, which means one straight or branched carbon chain of up to 20 carbon atoms which may be substituted by one or more -OH, -COOH, SO ₃ -rpynn, and / or optionally be interrupted once or repeatedly by one -O-, -S-, -CO-, -CS, -CONH-, -NHCO-, -NHCSNH-, -SO ₂ -, PO ₄ - or -NH-groups or one an aryl ring, and η stands for 1 to 5. Antibody and dye conjugates according to claim 1, characterized in that antibodies L ₁₉ and C. are used as antibodies. Antibody and dye conjugates according to claims 1 or 2, characterized in that the dye induces an optical signal in the visible spectral region of light. Antibody and dye conjugates according to claims 1 or 2, characterized in that the dye induces a fluorescence signal using a wavelength from a particular region of visible or near infrared light. A pharmaceutical agent comprising one or more conjugates of antibodies and dyes according to claims 1 to 4 for intraoperative imaging of the border regions (ie dimensions) of the outbreak of the disease. The pharmaceutical agent of claim 5, in admixture with suitable solvents, buffers and / or carriers. Use of antibody and dye conjugates and agents according to claims 1 to 6 for intraoperative imaging of the outbreak. Use of antibody and dye conjugates according to claims 1 to 6 for intraoperative imaging of the border regions of the outbreak. Use of conjugates of antibodies and dyes according to claims 1 to 6 for micro and macroscopic, intraoperative imaging of the border regions of the outbreak. Use of antibody and dye conjugates according to claims 1 to 4 for the preparation of a surgical treatment for angiogenesis-related diseases such as malignant tumors and their metastases, benign tumors, precancerous tissue changes, endometriosis, haemangiomas and haemangiomas .