WO2005000349A2

WO2005000349A2 - Passive immune therapy against malignant melanoma

Info

Publication number: WO2005000349A2
Application number: PCT/EP2004/006519
Authority: WO
Inventors: Ursula Wiedermann; Soldano Perrone; Hubert Pehamberger; Otto Scheiner; Erika Jensen-Jarolim; Christoph Zielinski; Heimo Breiteneder
Original assignee: Biolife Science Forschungs- Und Entwicklungsgesellschaft M.B.H.
Priority date: 2003-06-23
Filing date: 2004-06-17
Publication date: 2005-01-06
Also published as: EP1638604A2; DE10328121A1; CA2530655A1; WO2005000349A3; IL172751A0; AU2004251010A1

Abstract

Disclosed is the use of antibodies for producing an immunizing vaccine against HMW-MAA on melanomas.

Description

Passive immunotherapy for malignant melanoma

description

The invention relates to the use of antibodies for the production of a vaccine for immunization against malignant melanoma, in particular for passive immunization against high molecular weight melanoma associated antigen (HMW-MAA) - an important antigen - on melanoma, and the combination of antibodies and active Substances for the manufacture of a vaccine for the treatment of malignant melanoma.

Antigens can either be foreign to the body or the body's own (self-antigens) substances. In the case of foreign antigens - mostly proteins or polysaccharides - these are recognized by the immune system and, if they are classified as dangerous, eliminated by an appropriate immune response. The immune system, which has a non-specific and a specific branch of action, acts by means of humoral and cellular factors. This specific cellular defense is primarily carried out via cytotoxic T cells, which either directly dissolve a disease-causing agent or cells that are infected with pathogenic antigens, or the cells act via certain messenger substances that help other cells, the disease-causing substance or the disease-causing agent To eliminate condition.

The antibodies that are produced by B cells or plasma cells are counted among the humoral factors of the specific immune system. These antibodies are produced and secreted with the help of certain messenger substances, which in turn come from T cells (T helper cells). The antibodies formed are specific for a particular section on the surface of an antigen called a B cell epitope (there are also T cell epitopes, which are mostly linear and do not have the same location as the B cell epitopes on the molecular surface). There are several ways how an antibody can act: In principle, an antibody binds with its specific binding sites, the idiotopes / paratopes, in the hypervariable region of the Fab part of the antibody are located in specific regions of the antigen called epitopes - this binding takes place according to the "key and lock principle".

Through this binding, an antigen can now be neutralized and rendered harmless; in the case of a proliferating cell, the growth of the cell can be stopped (or increased) by the antibody binding; the bound antibody makes it easier for the antigen to be taken up by cells (opsonization and phagocytosis by means of appropriate "eating cells" - e.g. macrophages); or by binding the antibody with its Fc part to effector cells (macrophages, NK cells), these cells can be activated in order to be able to attack and eliminate the target cells or target organisms more easily.

In contrast to the foreign antigens, the body's own substances, cells etc. are not considered foreign and dangerous. On the contrary, it is even desirable that these self-antigens be tolerated by the immune system, since otherwise the inflammation and tissue damage would result constantly from the activation of the immune system (an example of a misdirection of the immune system towards self-antigens are autoimmune diseases).

If - due to a misdirection or failure of the regulatory system - there is excessive growth of the body's own cells, as is the case with a tumor, then the immune system usually does not respond because it has built up a tolerance towards the body's own proteins. On the basis of this fact, the tumor can spread freely in the body.

With the help of specific immunizations, a state of protection against certain pathogens or pathogenic agents can be achieved. There are two types of specific immunotherapy / immunization: active immunization - synonymous with vaccination - whereby substances (weakened pathogens, toxins, etc.) are supplied to the body against which the body itself builds protection. It takes several weeks to achieve the state of protection, the protection can last for several years, in some cases even for life. This differs from passive immunization (which is not a vaccination). Antibodies that have already been formed are supplied to the body, protection begins immediately with application and does not last very long because the antibodies are broken down. The protective state can be maintained for a long time by repeated administration of the antibodies.

In the case of the treatment of tumors, it is possible to achieve a reduction in tumor growth with the aid of passive immunization with antibodies which are directed against a specific tower origen. This could be the case in the treatment of breast cancer with the application of the antibody trastuzumab in combination with a standard chemotherapeutic agent (McLaughlin, P. et al. J. Clin. Oncol. 16, 2825-2833 1898), or rituximab for the treatment of relapses of non -Hodgkin lymphomas (Baselga, J., Norton, L et al. Cancer Research 58, 2825-2831 (1998)). The antibodies are humanized mouse antibodies (i.e. mice are immunized with human tumor cells and form specific antibodies against these foreign antigens; by exchanging the murine for a human Fc part using molecular biological methods, the humanized antibody is more tolerable for humans).

Melanoma belongs to a group of very malignant, often therapy-resistant tumors, which can occur in 90% of cases without genetic predisposition and in 10% with familial clustering. The incidence of malignant melanoma has increased enormously in recent years.

Only a stage I / II primary tumor with no breakthrough through the basement membrane can be cured by surgical removal in 85% of cases. In cases where the tumor growth is> 1.5 mm or lymph nodes are already affected (stages III / IV), the prognosis is very poor, e.g. This is because there is only an unsatisfactorily low percentage (approx. 20%) of effective therapy in these stages.

It has been shown that the aggressive growth of this tumor is associated with abnormal protein expression. One of the most important of these melanoma antigens is the HMW-MAA (high molecular weight melanoma associated antigen), which is found on 90% of all primary and metastatic tumors. The HMW-MAA is particularly responsible for the rapid tumor growth and tumor invasiveness. Biochemical analyzes have shown that the HMW-MAA is a glyocrotein-proteogycan complex with a molecular size of> 450 kDa. This antigen shows a strong immunogenicity because it contains numerous epitopes against which various monoclonal mouse antibodies have been produced.

One of these monoclonal antibodies is the 225.28 S antibody, which was generated by immunizing BALB / c mice with the human melanoma cell line M21. The location of the epitope recognized by the 225.28 S antibody differs clearly from that recognized by other monoclonal antibodies. The bound antibody is not endocytosed and remains attached to the membrane of the tumor cells.

An F (ab) 2 fragment of 225.28 S, which was conjugated to ^99m Tc (Technetium), was used as an immunoconjugate (Technemab-Kl; Sorin Biomedica, Italy) for the diagnosis of melanoma: In the article "Effects of Diagnostic Application of Monoclonal Antibodyon Survival in Melanoma Patients "by H. Bender et al, pages 65-68, Hybridoma vo. 16, No. 1, 1997, Mary Ann Liebert, Inc., describes the coupling of the F (ab) 2 part of an anti-melanoma antibody to a radioactive substance (technetium), this radioactively labeled antibody part for the diagnosis of melanoma or is used to diagnose the course of the tumor. It was found that people with whom the labeled antibody part was used had a longer survival time. It was also shown that the tumor-reducing effect is mostly due to the radio conjugate used.

However, there is a great concern to be able to do without radioconjugates when treating patients. It is therefore of great interest to achieve passive immunization, if possible without using conjugates. The object of the present invention is therefore to find a treatment route which is suitable for reducing or preventing the growth of melanoma and the formation of metastases, it being possible to largely dispense with radioconjugates.

The invention is based on the finding that antibodies are used which are suitable for passive immunization against malignant melanoma.

The invention is therefore to use antibodies which are suitable for producing a vaccine for passive immunization against the high molecular weight Melanoma Associated Antigen on malignant melanomas.

The High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) is found on 90% of all primary and metastatic tumors and is one of the most important melanoma antigens, as this antigen is associated with tumor spread and invasiveness. The use of antibodies directed against HMW-MAA is a particularly effective immunization, since this antigen is found in high numbers on malignant melanomas. It is particularly preferred that the antibodies used to produce a vaccine for passive immunization against HMW-MAA on malignant melanomas are monoclonal antibodies.

It is further preferred that the antibody is the monoclonal antibody 225.28S. This monoclonal antibody is generated by immunization (BALB / c-) mice with human Melanoma M21 cells. The monoclonal antibody 225.28S reacts strongly with the melanoma cells that express the HMW-MAA. For this reason, the monoclonal antibody 225.28S is particularly suitable for passive immunization against HMW-MAA on malignant melanomas. It could be shown by experiments that the monoclonal antibody 225.28S strengthens the tumor growth of melanomas in vivo. It can thus be seen from FIG. 2 that the growth of human tumor cells in SCID mice which are passively administered this antibody remains clearly behind compared to tumor cells in mice which were treated with control antibodies or saline. In this invention, it is described for the first time that intravenous applications of these antibodies lead to a massive tumor reduction. It should also be noted that this tumor reduction is achieved without the addition of a chemotherapeutic agent (as has been described for other monoclonal antibodies against, for example, breast cancer).

It is further preferred that the antibody is marker-free. Antibodies are mainly used to conjugate them with markers and then use these conjugates for diagnostic purposes in tumors. Fluorescence and / or radioactive markers are preferably used. The antibody in such a conjugate only serves to bring the active substance to the site of action and to bind it. In the present invention, however, it is preferred that the antibody itself serves as an active ingredient and thus acts directly against the HMW-MAA.

It is further preferred that the antibody is conjugate free, i.e. H. that the antibody is not linked to another substance. In the present invention, "connected" is understood to mean that they are covalent or ionic bonds or that there are other interactions, such as e.g. Hydrogen bonding or van der Walls forces. It is particularly preferred that the 225.28S monoclonal antibody is not conjugated.

It is further preferred that the entire antibody is used to prepare a vaccine for passive immunization against HMW-MAA on malignant melanomas. In particular, it is preferred that the whole antibody be the 225.28S monoclonal antibody.

Furthermore, it is also conceivable that fragments of antibodies can be used. It is particularly preferred that F (ab ') ₂ fragments of antibodies are used to prepare a vaccine for passive immunization against HMW-MAA on malignant melanomas. The F (ab) fragments are the parts of the molecule of the antibody which bind to the antigen (Fab- ^ fragment that binds the antigen). The prototype of the antibody has a symmetrical structure, consisting of four protein chains that are linked by non- covalent bonds and disulfite bridges are held together. After loosening these bonds, two chain pairs are formed, which are referred to as heavy (heavy or H) and light (light or L) chains due to their different molecular weights. However, if the antibody undergoes protolytic cleavage, three fragments are formed, two of which are composed of the L chain and the terminal ends of the H chain. These fragments are the F (ab) 'fragments mentioned above.

It is particularly preferred that F (ab ') ₂ fragments of the monoclonal antibody 225.28S are used.

It is further preferred that the antibody, in particular a monoclonal antibody and in particular the monoclonal antibody 225.28S thereof, with other active substances which act against HMW-MAA are used to produce a vaccine for passive immunization against HMW-MAA on malignant melanomas ,

The active substances are preferably chemotherapeutic agents. Chemotherapy cytostatics are particularly preferred, most preferred are dacarbine-DTIC, temozolamides, cisplatin, Fotemustine-muphoran and Vincristine-vinblastine.

In particular, it is preferred that the active substances are monoclonal antibodies and in turn are preferably antibodies that bind the HMW-MAA.

It is further preferred that these further monoclonal antibodies bind to other epitopes of the high molecular weight melanoma associated antigen than the monoclonal antibody 225.28S.

The invention is explained in more detail below by examples. Examples

animals

6-week-old pathogen-free C.BJ7 mice, obtained from Harlan Winckelmann Germany and kept in micro-isolator cages, received autoclaved food and water ad libitum during the experimental phase. All experiments were approved by the animal experimentation ethics committee of the University of Vienna and the Ministry of Development, Research and Culture.

Cell line and antibody

The human melanoma cell line 518 A2 (from Dr. Peter Schrier, Leiden, The Netherlands) was stored in a DMEM medium (life technologies, Carlsbad, CA) provided with 10% fetal calf serum and 1% antibiotics in a 5% C0 ₂ and 95 % humidified air atmosphere at 37 ° C. The cell culture was free of mycoplasma and pathogenic virus. The 225.28S monoclonal mouse antibody was provided by Soldano Ferrone. The mouse IgG monoclonal antibody clone LC1, which serves as a control antibody, was purchased from NeoMarkers, Fremont, CA.

Human melanoma SCID xenograft model

Experimental procedure (Fig. 1)

1 × 10 ^{7 of} the human melanoma cell line 518A2 resuspended in 200 μl sterile PBS were subcutaneously injected into the left flank of the mouse. Approximately The vaccine was administered intravenously 14 days later after the tumors reached an average diameter of 5 mm.

The mice were divided into three groups of 5 mice each. One group received the 225.28S monoclonal antibody, the second group received a control antibody (mouse IgG) and the third group only saline (untreated) was applied. 100 μg of the respective antibody (ie 5 mg / kg body weight) were administered intravenously in one Volume of 250 ul administered four times at 3 day intervals. The tumor size was determined twice a week by calibration measurement. The tumor volume was calculated using the following formula: Volume = (longest tumor diameter x shortest tumor diameter) / 2. The mice were sacrificed five days after the last injection. The tumors were exposed and their weight measured. The experiments were repeated three times under the same conditions.

Results

Reduction of tumor growth by administration of monoclonal antibody 225.28S in vivo.

In order to study the biological activity of 225.28S monoclonal antibodies in vivo, the monoclonal antibody was tested four times in an interval of three days in SCID mice (immune-incompetent mice lacking functional T and B lymphocytes) with an established human Melanoma administered. After three intravenous applications of monoclonal antibodies 225.28S, a significant reduction in tumor volume in the mice treated with monoclonal antibodies was found compared to the untreated mice (sham treated with sodium chloride solution) (FIG. 2). After the third antibody treatment, a lower tumor volume was also found compared to the mice that were treated with a non-specific control antibody. Five days after the last antibody treatment, the tumor volume of the mice treated with the monoclonal antibody was 50% lower compared to the mice treated with the control antibody and the untreated control mice (treated with sodium chloride).

At this point the tumor weight was also determined, which was also 50% lower than that of the control mice (FIG. 3). The experiments were carried out three times under the same conditions, the data shown representing the mean of the three test series. Statistk

The data from all three independent experiments were statistically evaluated using variance analysis and contrast analysis (* = P <0.05; * * = P <0.01). 5 mice / group are used per experiment.

Experimental procedure (see Fig. 1)

The experiment was started by subcutaneous injection of lxlO ⁷ melanone cells (518 A2) into the flank of the mice (dO). 14 days later, after the average diameter of the tumors had reached 5mm, the first intravenous injection of the monoclonal antibody 225.28S (100μg / 250μl) was injected, or the control antibody (100μg / 250μl) or the sodium chloride solution (sodium chloride, 250μl) was administered. The treatment was carried out four times in a three-day interval (dl4, 17, 20, 23) and then, after each antibody administration, the tumor size was measured. Five days after the last antibody administration, the mice were killed (d28) and the tumors exposed and weighed.

Tumor volume (see FIG. 2)

14 days after the inoculation of the tumor cells (dO) after the average diameter of the tumors had reached 5 mm, the first intravenous injection of the monoclonal antibody 225.28S (100 μg / 250 μl) or the injection of control antibodies (100 μg / 250 μl) or the injection of sodium chloride (Sodium chloride, 250μl). The treatment was carried out after 14, 17, 20 and 23 days. The mice were sacrificed five days later (d28). After each antibody application and at the end of the experiment, the tumor volume was evaluated. After the third application, a 50% reduction in tumor volume compared to the control antibody-treated or sodium chloride-treated mice could be determined (* = P <0.5; * * = P <0.01). Tumor weight (see FIG. 3)

After 28 days, the animals were sacrificed and the tumor exposed and weighed. Corresponding to the reduced tumor volume, the tumor weight after treatment with the monoclonal antibodies 225.28S was 50% lower than that of the control animals.

Claims

Expectations

1. Use of antibodies for the production of a vaccine for passive immunization against high molecular weight melanoma associated antigen on malignant melanoma.

2. Use according spoke 1 characterized in that the antibodies are monoclonal antibodies.

3. Use according to claim 2, characterized in that the antibodies 225.28S are monoclonal antibodies.

4. Use according to one of the preceding claims, characterized in that the antibodies are marker-free.

5. Use according to one of the preceding claims, characterized in that the antibodies are not conjugated.

6. Use according to any one of the preceding claims, characterized in that whole antibodies are used.

7. Use according to any one of claims 1-5, characterized in that F (ab ') fragments are used.

8. Use according to one of the preceding claims, characterized in that the antibodies are used together with another active substance.

9. Use according spoke 8, characterized in that the active substance is a monoclonal antibody, preferably an antibody against high molecular weight Melanoma Associated Antigen.

10. Use according to any one of claims 8-10, characterized in that the active substance contains a radioactive component.

11. Use according to any one of claims 8-10, characterized in that the active substance is a cytostatic.