SK6542002A3 - The use of antibodies for the production of pharmaceutical composition useful as vaccines - Google Patents

Abstract

The invention relates to the use of antibodies, which bind to antibodies for tumour-associated antigens and which are obtained from individual body fluids by immuno-affinity purification with specific ligands, for the production of individual, autologous, prophylactic or therapeutic vaccination against cancerous disease states. The ligands for immuno-affinity purification are antibodies or derivatives thereof, to tumour associated antigens. The invention further relates to pharmaceutical compositions, which comprise the antibodies produced by immuno-affinity purification, or the dendritic cells pulsed in-vitro by said antibodies.

Description

The present invention describes the use of antibodies that bind to antibodies directed against tumor-associated antigens ("tumourassociated antigens") and which are obtained from individual's body fluids containing antibodies by immunoaffinity purification on specific ligands for the manufacture of a pharmaceutical composition for an individual autologous prophylactic or therapeutic vaccination against cancer. Ligands for immunoaffinity purification are antibodies or derivatives thereof that are directed against one or more tumor-associated antigens. Furthermore, the invention relates to pharmaceutical compositions comprising antibodies obtained by immunoaffinity purification or dendritic cells which have been pulsed with them in vitro.

BACKGROUND OF THE INVENTION

The adaptive human immune system consists of two basic components, namely humoral and cellular immunity. The adaptive immune response is based on clonal selection of B and T lymphocytes, and in principle allows any antigen to be recognized and immunological memory created. Vaccination uses these properties of the adaptive immune system.

Each B cell produces an antibody with some binding specificity. This antibody is also present as a specific receptor in the membrane of those B cells that produce it. The humoral immune response against antigens which are recognized as foreign antigens is based on the selective activation of B lymphocytes that produce antibodies capable of binding to the epitopes of the antigen in question. During the differentiation of B lymphocytes is

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31938 / T reorganization (remodeling) of DNA by a key process in terms of antibody diversity.

A large number of antibodies of different specificities, isotypes and subclasses are present in human serum. The total serum concentration of all immunoglobulins is 15 to 20 mg / ml, which means that approximately 100 g of immunoglobulins of varying specificity circulate continuously in the bloodstream. It is not possible to determine the exact amount of all antibodies having different specificities. Theoretically possible repertoire is approximately 10 ¹¹ . Generally, an antibody may bind different antigens that are similar to each other but bind them with different affinities and avidities.

The immune system must maintain homoeostasis in terms of distribution and importance of these different specificities through endogenous regulatory mechanisms. The essential mechanism is the so-called. "Idiotypic network", which was postulated in Niels Jerna about 25 years ago (Jern, Ann. Immunol. 125C (1974), 373-389): There are anti-idiotypic antibodies that are directed against each of the antibody idiotypes that determine its binding specificity and which therefore bind to the idiotype of the first antibody as a means of antigen recognition. Jern assumed that interactions between idiotypic specific receptors on lymphocytes may be responsible for regulating the immune system. Obviously, these interactions exist because it has been shown that anti-idiotypic antibodies directed against primarily induced antibodies have also been produced during the immune response. Because there are anti-idiotypic antibodies against each antibody, lymphocytes are in principle not tolerant of antibody idiotypes.

In an immunological sense, some anti-idiotypic antibodies may represent an "internal image of an antigen." Thus, such antibodies can be used for immunization as a replacement for the nominal antigen, since antibodies induced by immunization can, at least in part, bind to the nominal antigen. After a considerable period of time, this approach has been used, especially in tumor immunotherapy. For more than 10 years, a number of preclinical and

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31938 / T clinical projects on how to induce immune responses against tumor-associated antigens by monoclonal anti-idiotypic antibody vaccination (for review see: Bhattacharya-Chatterjee, Foon, Anti-idiotype antibody vaccine therapies of cancer, Cancer Treat. Res. 94 (1998), 51-68. Most of these monoclonal anti-idiotypic antibodies are of murine origin. However, human monoclonal anti-idiotypic antibodies have also been tested (e.g., Fagerberg et al., PNAS 92 (1995), 4773-4777).

A particular case is the attempt to therapeutically immunize patients suffering from B cell lymphoma. In the case of such a disease, a particular B cell clone that forms specific immunoglobulins having its idiotypic characteristics, and which also carries a given immunoglobulin as a receptor in the cell membrane, is degenerate. This antibody is monoclonal and can be considered a tumor-specific antigen for individual B cell lymphoma. It has been shown that such patient-specific autologous antibodies, when produced in cell culture and administered as a vaccine in a suitable immunogenic form, can elicit an immune response against the idiotype of such an antibody, which immune response affects B cell lymphoma (Reichardt et al. Blood, 93 (1999), 2411-2419). In short, that

Human monoclonal anti-idiotypic antibodies that mimic (mimic) a tumor-associated antigen can, as a vaccine, induce an immune response in cancer in patients, wherein the immune response can be directed against a given tumor-associated antigen. Using hybridoma technology or immortalization of human B cells, such human monoclonal antibodies were obtained from patients who received (mostly murine) anti-tumor antibodies for passive immunotherapy and developed an immune response against them. Other patients were immunized with human monoclonal anti-idiotypic antibodies generated in this manner.

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Human antibodies that have been produced by B-cell lymphomas and thus have a defined idiotype can, as individual vaccines, induce an immune response in a patient-specific manner when the immune response can be directed against B-cell lymphoma. Such patient-specific antibodies must be obtained individually, using hybridoma technology or by immortalizing cells from B cell lymphoma. The use of such antibodies is naturally limited to the treatment of B cell lymphomas.

Thus, although it is known that monoclonal anti-idiotypic antibodies from certain biological species can also be used to immunize individuals of the same species from which the antibodies were derived, it is in any case necessary to manipulate and cultivate the cells. Thus, this approach is considerably time consuming and is limited to monoclonal antibodies only.

Losman et al., Proc. Natl. Acad. Sci. USA 88 (1991), 34213425, the purification of ab2 antibodies from a patient's carcinoma has been described. However, this patient was previously treated with a mouse monoclonal antibody (NP4) against CEA. Thus, it is clear that there is a continuing need for means and methods of treatment that would allow for effective and widely applicable treatment, and optionally individual (i.e., individualized) treatment of tumors as well as prevention of tumor diseases.

Thus, the technical problem addressed by the present invention is to provide effective means for individually treating various types of tumors, generally for the treatment of cancer, and also for preventive protection against cancer. This technical problem has been solved as demonstrated by the exemplary embodiments of the invention defined by the appended claims.

The present invention relates to the use of antibodies for the manufacture of compositions useful as vaccines for both prophylactic and therapeutic vaccination against cancer, wherein the antibodies are obtained from body fluids containing the antibodies by immunoaffinity purification, wherein the antibodies recognize one or more antigens associated with tumors or their

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319387 fragments having the same idiotype are used as ligands for immunoaffinity purification.

The pool of antibodies, for example, present in large amounts in each human's blood, contains antibodies with all possible binding specificities. Thus, this antibody pool also essentially comprises antibodies (Ab2) against an idiotype e.g. (already known or also new) murine monoclonal antibodies (Ab1) directed against any tumor associated antigen (TAA). In accordance with immunological network theory, the same antibody pool of each human also contains a certain amount of antibodies directed against the idiotypes of autologous Ab2, these antibodies being termed Ab3 antibodies. According to immunological network theory, Ab3 antibodies can bind to a tumor-associated antigen that is defined by external Ab1. Shifting the Ab2 / Ab3 balance in favor of Ab3 must result in the immune system being able to better recognize and then attack tumor cells bearing the antigen.

It is an object of the present invention that such a shift in immunological balance can be achieved by administering individual autologous fractions of Ab2 in the form of an immunogenic vaccine. By such autologous vaccination, B cells that form Ab3 can be selectively stimulated. For such vaccination, only small amounts of the Ab2 fraction are needed, which can be formulated by known methods using a suitable vaccine adjuvant.

Thus, the antibodies used according to the invention are Ab2 antibodies of body fluids containing antibodies that are directed against the anti-TAA antibody used for immunoaffinity purification. Body fluids containing antibodies are, for example, blood, plasma, serum, lymph, malignant discharge, and the like. The preferred body fluid is serum. Body fluid can be obtained from any animal organism that contains body fluids containing antibodies. They are preferably vertebrate body fluids, more preferably mammals, and most preferably human.

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Since the immune response induced by vaccination with autologous antibodies is determined by the binding region of these antibodies, i.e. their idiotype, in principle fragments or derivatives of these antibodies may also be used instead of the intact antibody fraction, if these fragments or derivatives contain an idiotype corresponding to the starting antibody. Thus, the term "antibody" in the present specification also includes fragments or derivatives of such antibodies having the same binding specificity. Non-limiting examples include the following: F (ab) ' ₂ fragments and F (ab)' fragments, which can be prepared by biochemical methods known (e.g., by enzymatic cleavage). The term "derivative" also includes e.g. antibody derivatives that can be prepared by chemical or biochemical methods known in the art, such as antibodies amidated by fatty acids on the free amino group to enhance lipophilic character, allowing incorporation into liposomes. In particular, the term "derivative" also includes products that can be prepared by chemical condensation of an antibody or antibody fragment with a molecule that enhances an immune response, such as tetanus toxoid, Pseudomonas exotoxin, lipid A derivatives, GM-CSF, IL-2, or by chemical condensation of the antibody or antibody fragment with polypeptides that enhance the immune response, such as GM-CSF, IL-2, IL-12, C3d, etc. Purification of antibodies from antibody-containing body fluids, e.g. from human serum, using immunoaffinity purification, is performed according to procedures known to those skilled in the art (Clin. Chem. 45 (1999), 593, J. Chem. Technol. Biotechnol. 48 (1990), 105). The TAA-specific antibody or antibody mixture is immobilized on a solid phase (carrier). It may be a membrane, gel or the like to which the antibodies can bind without substantially altering their binding properties. The Ab2 may be bound to the immobilized antibodies in a batch or flow manner. Immunoaffinity purification can be performed automatically on a chromatographic apparatus by hand. But one can even imagine that

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The 31938 / T process is performed manually, automatically, or semi-automatically using a simple device containing immobilized antibodies. The immunoaffinity chromatography described in Example 1 usually yields between 3 and 10 µg of immunoglobulin per ml of serum used. The general antibody fraction obtained in this way contains both IgM and IgG. Thus, it is possible to isolate about 0.08 to 0.25 mg of Ab2 from the amount of blood that can be conveniently collected, e.g. of 50 ml of blood to give approximately 25 ml of serum. This amount is substantially sufficient for vaccination. If it is necessary to isolate a greater amount of Ab2, techniques known to those skilled in the art, such as plasmapheresis, may be used in combination with immunoaffinity purification. When several antibodies having different specificities are used, such immobilized antibodies can be used simultaneously or separately and immunoaffinity purifications can be performed in parallel or in series.

In the context of the present invention, the term " tumor associated antigen " refers to a structure that is presented primarily by tumor cells and thus allows them to be distinguished from normal non-tumor cells. Preferably, such a tumor associated antigen is located on or in the cell membrane of the tumor cells. However, it is not excluded that such antigens are also present on non-degenerate cells. Tumor-associated antigens are, for example, polypeptides, particularly glycosylated proteins or glycosylation profiles of polypeptides. Other structures that may represent a tumor-associated antigen are, for example, glycolipids. These include, for example, gangliosides such as GM2. Furthermore, a tumor-associated antigen may be represented by a change in the cell membrane lipid composition that may be characteristic of cancer cells.

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Various antibodies to various tumor-associated antigens have been known for a long time, or can be prepared by methods known to those skilled in the art (e.g., hybridoma, phage display, phage display or phage display) techniques.

In a preferred embodiment of the invention, the antibodies obtained by immunoaffinity purification are administered as autologous, individual vaccines to individuals from the body fluids from which they have been obtained.

Thus, another indivisible component of the present invention is that isolating the Ab2 fraction from the body's fluids, e.g. blood, may be performed by immunoaffinity purification of the antibody present in the body fluid. One or more antibodies against the tumor associated antigen are used as ligand.

In this context, the subject is any animal, preferably a vertebrate, more preferably a mammal, and most preferably a human.

In another preferred embodiment, several antibodies directed against different tumor-associated antigens and / or different epitopes of one or more tumor-associated antigens can be used simultaneously for immunoaffinity purification. When several, and in particular monoclonal, antibodies directed against different tumor-associated antigens or different epitopes are administered simultaneously for immunoaffinity purification of Ab2 present in a body fluid, a fraction containing different Ab2 is obtained against a selected set of tumor-associated antigens, simultaneous use as a "vaccine" trigger for an immune response against all these tumor or epitope associated antigens. Thus, the immunological equilibrium is shifted one step towards the recognition of a set of (often co-expressed, co-expressed) tumor-associated antigens or epitopes. Such a procedure naturally increases the efficacy of the induced immune response and substantially reduces generation

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31938 / T antigen-negative tumor variants ("tumor leakage") because it is highly unlikely that tumor cells could stop the expression of several antigens simultaneously.

In principle, the methods described above are based on all known or newly discovered tumor-associated antigens. The only condition is that one or more antibodies, preferably monoclonal antibodies directed against this antigen are available and can be administered as a ligand for immunoaffinity purification. In principle, all possible types of antibodies are suitable, in particular polyclonal or monoclonal antibodies, with monoclonal antibodies being preferred. It is also possible to use mixtures of polyclonal and monoclonal antibodies. In addition, it can be applied as e.g. a mouse monoclonal antibody as well as an antibody from another biological species (e.g., a rat). In addition, murine-human chimeric antibodies, humanized or human monoclonal antibodies against tumor-associated antigens may be administered. The properties of the antibody used for immunoaffinity purification are determined by their binding regions, i. their idiotype. Thus, in principle, fragments of these antibodies can also be used for successful immunoaffinity purification instead of an intact antibody, as long as the fragments still retain the idiotype of the respective starting antibody. Examples include, but are not limited to: F (ab) '2 fragments, F (ab)' fragments, and Fv fragments, which can be prepared by biochemical methods known to those skilled in the art (enzymatic cleavage) or molecular biology methods , also known to those skilled in the art. As a matter of fact, mixtures of said antibody species or fragments thereof can also be used.

The choice of tumor-associated antigens and therefore the antibody used for immunoaffinity purification that is directed against such antigens depends on the antigenic properties of the tumor against which the vaccination is to be carried out.

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As an example, tumor associated antigens known in the art and which are often expressed on various, especially epithelial, tumors are given as an example. However, the use of the autologous vaccination described in the present application is not at all limited to the following antigens:

- Epithelial cell adhesion molecule (Ep-CAM)

- Carcinoembryonic antigen (CEA)

- Lewis Y carbohydrate,

- sialyl Tn saccharide,

- globo H saccharide,

gangliosides such as GD2 / GD3 / GM2

- Prostate-specific antigen (PSA)

-CA 125,

-CA18-9.

-CA 15-3,

- TAG-72

- EGF receptor,

- Neuronal cell adhesion molecule (N-CAM)

- Her2 / Neu receptor,

-D97.

- D20 a

-CD21

In particular, monoclonal antibodies which are in principle suitable for use as ligands for higher immunoaffinity purifications in order to obtain Ab2 have been described against all of these antigens (mostly several).

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Other tumor-associated antigens have been described, for example, in DeVita et al. (Eds., &Quot; Biological Therapy of Cancer ", 2 ^nd edition, chapter 3: Biology of Tumor Antigens, Lippincott Company, ISBN 0-3975144116-6 (1995)).

In a preferred embodiment of the invention, the tumor associated antigen in the membrane is a localized molecule that is often expressed or coexpressed on tumor cells of epithelial origin, tumor cells of neuroendocrine origin or tumor cells derived from a hematopoietic system.

In another preferred embodiment, the composition of the invention is suitable for repeated administration of an antibody contained therein. Method. obtaining an autologous vaccine from an individual's body fluids is not naturally limited to a single application.

The shift of the immunological repertoire towards anti-tumor efficacy that is triggered by the first vaccination may be enhanced by repeating the same procedure, e.g. several weeks after isolation of the first autologous vaccine by immunoaffinity purification, again, body fluid, e.g. blood, and a new autologous vaccine can be prepared and administered. In this way, it is ensured that the respective immunological equilibrium state is always taken into account for individual vaccines. This procedure may be repeated at appropriate intervals (e.g., at least every 4 to 8 weeks, then every 6 months).

The novel vaccine compositions and methods of autologous antibody vaccination described in the present application are in principle suitable for both therapeutic and prophylactic purposes. Repeated therapeutic vaccination of cancer patients may inhibit the formation of new metastases, which may at least slow the progression and spread of the disease. In particular, therapeutic autologous vaccination may be useful in the following stages of the disease:

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In the early stages of the disease, e.g. following successful primary tumor surgery (adjuvant stage), where the remaining disseminating tumor cells are destroyed by the autologous vaccine of the invention, thereby preventing the emergence of new metastases. As a result, the relapse-free life and thus the survival time may be prolonged. Alternatively, lifetime protection against metastasis can be achieved by the described autologous vaccination and boosters at preferred intervals.

At the stage when the metastases have already been formed, their spread and the formation of further metastases can be stopped by the formulations or autologous vaccination of the invention. As a result, the condition of the disease stabilizes and thus it is possible to maintain the quality of life and possibly extend the life expectancy.

The autologous vaccination strategy of the invention can be used in patients who have been given a monoclonal antibody against a tumor-associated antigen in advance for diagnosis or therapy and who have developed an immunological response against them. As a result, in immunoaffinity purification in which this antibody is used as a ligand, antibodies directed against it (Ab2) are obtained in an amount higher than that of untreated individuals. Vaccination with these Ab2 antibodies enhances the production of Ab3 antibodies that may already have been produced internally. Intrinsic (intrinsic) induction of Ab3 antibodies with potential antitumor properties by induction of Ab2 using passive immunotherapy with murine antibodies directed against tumor-associated antigens (Ab1) that are administered i.v. cancer patients, has been predicted and tested for many years (e.g., Koprowski et al., PNAS 81 (1984), 216-19).

The composition of the invention and the autologous vaccination of the invention can, in principle, be used in healthy individuals to improve, i.e., prophylaxis, the protection against cancer. Such measures make sense, especially in the case of high-risk groups (where individuals belong, for example, with a genetic predisposition for the development of a particular cancer that may be

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31938 / T detected to an increased extent by corresponding tests).

The fact that the immunological condition of an individual with respect to an idiotypic network is taken into account is because the preferred vaccine is prepared from the individual's body fluid, e.g. serum is generally an advantage of the individual autologous vaccination strategy described in the present invention.

Furthermore, the consequence is that the immunized individual does not come into contact with foreign antigens, but is only subjected to an endogenous component in a suitable form that modulates the immunological balance.

In a preferred embodiment of the invention, the antibody obtained by immunoaffinity purification is formulated with an adjuvant suitable for vaccines.

As generally applicable to vaccines, the fraction of the autologous antibody or fragments and derivatives thereof may be formulated with vaccine adjuvants. The adjuvant enhances the immune response. Examples of such adjuvants include, but are not limited to: aluminum-containing adjuvants, particularly aluminum hydroxide (e.g., Al-gel), lipopolysaccharide derivatives, Bacillus Calmette Guerin (BCG), QS-21, liposome formulations, formulations with other antigens against which the immune system already has a strong immune response, such as tetanus toxoid or influenza virus components, optionally in the form of liposome preparations.

To enhance the immune response, the obtained vaccine may be administered together with the corresponding, preferably human, cytokines that promote the development of an immune response. In particular, but never exclusively, it is necessary to mention the granulocyte and macrophage colony stimulating factor (GM-CSF). This cytokine stimulates an effective immune response by activating antigen processing cells, e.g. dendritic cells.

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Alternatively, fractions of the autologous antibody can be incubated with the autologous dendritic cells cultured ex-vivo by methods known to those skilled in the art. The dendritic cells thus pulsed are then administered back to the subject. In this way, particularly effective immune responses can be achieved.

In a preferred embodiment of the use according to the invention, the antibodies contained in the composition are mixed with an adjuvant and are then heat treated, preferably at a temperature between 70 and 121 ° C. The adjuvant is preferably an aluminum-containing adjuvant. It is possible that although heat treatment denature the protein antigen, the immunogenic portions of the protein may be presented to the immune system in the proper form by binding to the adjuvant. However, it is not absolutely necessary to denature the proteins in order to obtain the beneficial effect of heat. It is known that thermal denaturation depends not only on the temperature itself, but also on the time the protein is exposed to that temperature. In addition, there are other physicochemical parameters such as ionic strength, pH, or the type and amount of active surface structures in the composition that are responsible for protein denaturation. Thus, conditions may occur where the antibodies are not or are not fully denatured and / or other effects, such as less desorption at the surface of the adjuvant, are utilized.

A further advantage of formulating the vaccine together with the adjuvant and subsequent heat treatment is that in such a formulation, the infectious pathogens are optionally attenuated or completely inactivated. This advantage may be important in both production and storage and distribution of the vaccine formulation. Thus, there is considerable safety with respect to both known and unknown pathogens of infectious diseases. In addition, it is possible to fill medicaments without the use of preservatives, if the appropriate packaging method is used, since the heat treatment has provided antimicrobial preservation of the vaccine.

A further advantage of the formulations thus formulated is the potential to increase the immunogenicity of the antibody, since heating may result in at least partial denaturation of the antibody. Such increased antigenicity may result in increased immunogenicity, particularly in the case of proteins that could be

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31938 / T recognized as a body's own protein. Yet another advantage is that the anti-adjuvant complex is potentially additionally stabilized by heat inactivation, i. Desorption of the protein antigen does not take place as quickly as is the case with the preparation containing the antigen and the adjuvant but not heat-treated. This advantage also allows for longer intervals between immunizations.

The composition produced according to the invention may be administered in accordance with generally known procedures, e.g. as a vaccine by subcutaneous or intramuscular injection.

Furthermore, the present invention relates to a pharmaceutical composition comprising antibodies isolated from body fluids containing antibodies by immunoaffinity purification, wherein for immunoaffinity purification, antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype are used as ligands. As mentioned above, such formulations are useful as vaccines for therapeutic or prophylactic vaccination against cancer. With respect to preferred embodiments of the formulation, for ingredients, formulations, routes of administration, etc. The pharmaceutical composition of the invention is as previously described in connection with the use of the invention.

Finally, the present invention also relates to a pharmaceutical composition comprising isolated dendritic cells of an individual that have been cultured ex vivo with antibodies that have been isolated from a body fluid containing antibodies of the same individual by immunoaffinity purification.

In addition, the present invention also relates to therapeutic or prophylactic cancer vaccination when administering to an individual an antibody that has been isolated from a body fluid containing antibodies, preferably from the body fluid of the same individual, by immunoaffinity purification, wherein antibodies are used for immunoaffinity purification as ligands. recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype.

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In addition, the present invention relates to therapeutic or prophylactic vaccination against cancer, wherein an individual is administered autologically isolated dendritic cells that have been cultured ex vivo and which have been previously incubated in vitro with antibodies that have been isolated from a body fluid containing antibodies, preferably body fluid. of an individual, by immunoaffinity purification, wherein for immunoaffinity purification, antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype are used as ligands. Essentially the same as previously explained in connection with the use according to the invention also applies to other preferred embodiments.

The present invention further relates to a method of making antibody preparations, wherein the antibodies are antibodies that have been isolated from a body fluid containing immune purification antibodies, wherein antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype are used as ligands. With respect to body fluids, the use of antibodies as ligands, and tumor-associated antigens, essentially the same applies as previously explained in connection with the use of the invention. Immunoaffinity purification can be accomplished by methods known to those skilled in the art. In this respect, the same applies as already mentioned above.

In a preferred embodiment of the method of the invention, several antibodies directed against different tumor-associated antigens and / or epitopes of such antigens are used simultaneously for affinity purification. In a procedure where several antibodies are used for affinity purification, they can be used either simultaneously or separately and immunoaffinity purifications are performed in parallel or serially, i. different epitopes and / or antigen specificities may be combined as desired. These requirements are due to the expression of different antigens on tumors of different individuals.

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In a particularly preferred embodiment of the invention, monoclonal antibodies are used as ligands for immunoaffinity purification, and in an even more preferred embodiment serum is used as body fluid.

The method of the invention is preferably a method of preparing an antibody preparation, which is further used to produce a pharmaceutical preparation that is useful for therapeutic or prophylactic vaccination against cancer.

Furthermore, the present invention relates to a process for the manufacture of a pharmaceutical composition, wherein the antibody is prepared by the method of the invention and the antibody thus obtained is subsequently formulated with a pharmaceutically acceptable carrier and / or a suitable vaccine adjuvant.

In a preferred embodiment, the antibody preparation is admixed with an adjuvant, preferably an aluminum-containing adjuvant, and then subjected to heat treatment preferably at a temperature between 70 and 121 ° C.

Also for these preferred embodiments of the pharmaceutical formulations of the invention is all that has been stated above.

All patents, publications or database accesses cited in this application are incorporated by reference in their entirety. application.

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BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a chromatogram of immunoaffinity purification (using immobilized 17-1A antibody) of the antibody fraction from rhesus sera.

Figure 2 shows a chromatogram (sorting chromatography) of the antibody fraction purified by immunoaffinity purification.

Figure 3 shows the binding of serum antibody obtained by affinity purification to antibody 17-1 A: 17-1 A ELISA.

Figure 4 shows the result of autologous vaccination of rhesus macaque: binding of serum Ig to Kato III tumor cells (cell ELISA).

Figure 5 shows a chromatogram (sorting chromatography) of the antibody fraction purified by immunoaffinity purification (antiEpCAM / anti-Lewisy mixed beds).

Figure 6 shows a size standard chromatogram (separated by sorting chromatography).

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DETAILED DESCRIPTION OF THE INVENTION

The following materials were used in the examples, which are further described to further illustrate the present invention. The examples are not intended to limit the scope of the invention in any way. ^!

Microtiter plates: F96 MaxiSorp (Nunc) Immuno Plates for ELISA, Cell Culture Cluster Cell Cells (Costar, Catalog # 3598) for ELISA with cells

Cell lines: KATO III: human gastric carcinoma cell line (ATCC

HTB 103)

Condensation buffer: 0.1 M NaHCO ₃ 0.5 M NaCl pH 8.0 Purification buffer A: PBS def. 0.2 M NaCl Purification buffer B: 0.1 Mglycine / HCl 0.2 M NaCl pH 2.9 Medium A: RPMI 1640 + 2 g / l NaHCO ₃ 100 U / ml penicillin G 100 pg / ml streptomycin sulfate 4 mM glutamine 10% fetal calf serum (heat inactivated) Coupling buffer: ¹ 15 mM Na ₂ CO ₃ 35 mM _NaHCO3 3 mM _NaN3 pH: 9.6

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Deficient PBS (PBS def.): 138 mM NaCl

1.5 mM KH ₂ PO ₄

2.7 mM KCl

6.5 mM Na ₂ HPO ₄ pH: 7.2

Fixation solution: 0.1% glutardialdehyde in saline NaCl

Wash Buffer A: 2% NaCl 0.2% Triton X-100 in PBS def. Wash Buffer B: 0.05% Tween 20 in PBS def. Locking buffer A: 5% fetal calf serum (heat inactiv.) In PBS def. Blocking buffer B: 1% bovine serum albumin 0.1% NaN ₃ in PBS def. Dilution buffer A: 2% fetal calf serum (heat inactivated) in PBS def. Dilution buffer B: PBS def. Marking / dyeing buffer: 24.3 mM citric acid 51.4 mM Na ₂ HPO ₄ pH: 5.0

substrate: 40 mg o-phenylenediamine dihydrochloride 100 ml labeling / dyeing buffer 20 μΙ H ₂ O ₂ (30%)

Stop solution: 4 NH ₂ SO ₄

Formulation buffer: 10% PBS def., PH = 6.0% NaCl saline

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Example 1

Preparation of an immunoaffinity column with TAA-specific antibody

7.5 g of CH-Sepharose 4B (Pharmacia) was suspended for 15 minutes in 20 ml of 1 mM HCl. The resulting gel was then washed with 1 L of 1 mM HCl and then with 200 mL of condensation buffer on an AG3 sinter filter. 100 mg of mouse 17-1 A antibody (Panorex, 10 mg / ml stock solution, specific for the tumor associated antigen Ep-CAM) was dialyzed against 5 L of condensation buffer and adjusted with a condensation buffer to a final concentration of 5 mg / ml. This solution was mixed with the gel suspension in a sealed container. A suspension suitable for condensation may be prepared at a 1: 2 gel to buffer ratio. This suspension was poured for 24 hours at 4 ° C. Excess ligand was then eluted with 3 x 30 ml condensation buffer. The remaining reactive groups were blocked by 1 hour incubation with 1M ethanolamine at 4 ° C. Then the gel was rotated for 1 hour at room temperature with 0.1 M Tris-HCl buffer. Finally, the gel was washed in three cycles of pH-changing buffer. Each cycle consisted of 0.1 M acetate buffer (sodium acetate) pH 4 with 0.5 M NaCl, and 0.1 M Tris-HCl buffer pH 8 with 0.5 M NaCl. The gel was then stored at 4 ° C.

Example 2

Immunoaffinity purification of the antibody fraction from serum by immunoaffinity chromatography ml of peripheral blood was collected from rhesus monkeys and serum was prepared therefrom. The entire procedure was carried out under sterile conditions.

Immunoaffinity purification was performed on a FPLC system (Pharmacia). 1 ml of the gel prepared according to Example 1 was loaded onto an HR5 / 5 column (Pharmacia). 5 ml of serum was diluted 1:10 with Purification Buffer A. This solution was pumped through the column at a flow rate of 1 ml / minute and then the column was flushed with Purification Buffer A until it was reached again.

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31938 / T basal value of the UV detector (280 nm). Bound immunoglobulins were eluted with purification buffer B and the fraction was neutralized with 1 M Na ₂ HPO ₄ immediately after desorption. Figure 1 shows a chromatogram from this purification (UV 280 nm).

μΙ of the antibody fraction purified as described above was analyzed on a sorting column (SEC, Zorbax 250 GF). 220 mM phosphate buffer, pH 7 + 10% acetonitrile were used as carrier. As can be seen from the chromatogram of this analysis, (Figure 2), the antibody fraction contains approximately 3: 2 IgM and IgG. The total amount of antibody fraction was approximately 40 µg (determined by size exclusion chromatography (SEC)). The antibody fraction obtained in this way was tested in ELISA for binding to antibody 17-1A (which was used as a ligand for affinity purification): 100 μΙ aliquots of antibody specific to tumor-associated antigen (antibody 17-1A, solution with 10 g / g) ml in binding buffer), which was also used for affinity purification, were incubated in the wells of the microtiter plate for 1 hour at 37 ° C. After washing the plates six times with wash buffer A, 200 μΙ blocking buffer A was added and incubated for 30 minutes at 37 ° C. After washing the plates as described above, 100 µl aliquots of the affinity purified antibody fraction to be tested, together with normal human immunoglobulin at the same concentration as the negative control, were incubated at a dilution of 1: 4 to 1:65,000 in dilution buffer A for 1 hour at 37 ° C. After washing the plates again as described above, 100 μΙ of peroxidase-conjugated goat anti-human Ig antibody (Zymed) was added at a 1: 1000 dilution in dilution buffer A and incubated for 30 minutes at 37 ° C. Plates were washed four times with wash buffer A and twice with staining / staining buffer. Antibody binding was detected by adding 100 μΙ of specific substrate and the color reaction was stopped in approximately 3 minutes by adding 50 μΙ of stop solution. Evaluation was performed by measuring the optical density (OD) at a wavelength of 490 nm (the wavelength of the reference measurement was 620 nm).

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As can be seen in FIG. 3, the affinity purified antibody fraction showed significant binding to antibody 17-1 A, while normal human immunoglobulin did not bind at all.

I

Example 3

Vaccine formulation with purified antibody

The antibody fraction obtained by affinity purification was formulated with aluminum hydroxide as an adjuvant as follows:

mg of aluminum hydroxide (aqueous suspension, Alhydrogel, Superfos) was added to 3 ml of affinity chromatography solution (containing approximately 40 µg of antibody) and the suspension was centrifuged in a "FILTRON" centrifuge vial (Microsep ™, 10 kD filter limit) for 30 minutes at 4000 x g. Then the solution was suspended twice with 1 ml of formulation buffer and centrifuged for 30 minutes at 4000 x g. The suspension was made up to 0.5 ml with formulation buffer and stored in a sterile manner.

Example 4

Immunization of rhesus monkeys with an autologous immunoaffinity purified antibody

The respective rhesus monkeys from which the vaccine was obtained, as described in Examples 2 and 3, were vaccinated subcutaneously in the back with this vaccine. Prior to the first vaccination, 5 ml of blood was drawn for serum preparation (to establish baseline values to characterize the immune response). Two weeks thereafter, 10 ml of blood was collected, from which serum was again isolated. With 4 ml of this serum, autologous vaccine isolation was repeated as described above. Rhesus monkeys were injected subcutaneously in the back with this new vaccine. Four weeks after this vaccination, 5 ml of blood was collected again and serum was isolated (to determine the effect of vaccination).

The pre-serum of these macaques and the immune serum 4 weeks after the second

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The wells of the microtiter plate were incubated with a 100 μΙ cell suspension of the KATO III line at 2x10 ⁶ cells / ml in medium A overnight at +37 ° C. After aspiration of the supernatant, the plates were incubated with 50 μΙ fixation solution in each well for 5 minutes at room temperature. After aspirating the supernatant again, 200 µL of blocking buffer B was added to each well and the plates were incubated for 1 hour at room temperature. After washing twice with 200 μΙ wash buffer B, 100 μΙ aliquots of cynomolgus serum were tested by incubating at a dilution of 1: 4 to 1:56 000 with buffer B for 1 hour at 37 ° C. After washing the plates twice with 100 μΙ ice-cold wash buffer B, 100 μΙ of peroxidase-conjugated goat anti-human Ig antibody (Zymed) diluted 1: 1000 with buffer A was added and incubated for 45 minutes at 37 ° C. Plates were washed three times with 100 μΙ ice cold wash buffer B. Antibody binding was detected by the addition of 100 μΙ specific substrate and the color reaction was stopped after approximately 5 minutes by adding 50 μΙ of stop solution. Evaluation was performed by measuring the optical density (OD) at a wavelength of 490 nm (reference wavelength was 620 nm).

As can be seen in Figure 4, immunoglobulins that bind to Kato III tumor cells occur in immune sera of macaques vaccinated in an autologous manner, whereas such antibodies could not be detected in the pre-serum of the same animals.

The results of the experiments described above show that vaccination with individual fractions of autologous antibody obtained by immunoaffinity purification with a monoclonal antibody directed specifically against a tumor-associated antigen, triggers a humoral immune response that is directed against human tumor cells associated with the tumor antigen. .

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Example 5

Preparation of an immunoaffinity column with two different TAA-specific antibodies.

The monoclonal mouse antibody that reacted with EP-CAM and the monoclonal mouse antibody that reacted with Lewis Y were both coupled to activated CH sepharose 4B (Pharmacia Biotech, Sweden) by a standard procedure:

7.5 g of CH sepharose 4B was suspended in 20 ml of 1 mM HCl for 15 minutes and the soaked gel was washed with 1 liter of 1 mM HCl and then with 200 ml of condensation buffer on a sintered glass AG3 filter. 100 mg of mouse antibody (10 mg / ml stock solution) was dialyzed against 5 L of condensation buffer and then adjusted to 5 mg / ml with condensation buffer. This solution was mixed with the goal suspension in a sealed container. A suspension that was suitable for condensation was obtained at a gel to buffer ratio of 1: 2. The suspension was rotated at 4 ° C for 24 hours. The excess ligand was then washed with 3 x 30 ml of condensation buffer. The remaining reactive groups were blocked by 1 hour incubation with 1 M ethanolamine at 4 ° C. Then the gel was rotated for 1 hour at room temperature with 0.1 M Tris-HCl. Finally, the gel was washed in three cycles of pH changing buffer. Each cycle consisted of 0.1 M acetate buffer (sodium acetate) pH 4 with 0.5 M NaCl, followed by 0.1 M Tris-HCl pH 8 with 0.5 M NaCl. The gel was then stored at 4 ° C.

Two equal volumes of two affinity matrices were mixed to form an immunoaffinity chromatography column (Akta system, Pharmacia, Sweden).

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Example 6

Purification of monkey serum antibody by simultaneous immunoaffinity purification with two different TAA-specific antibodies.

Approximately 20 ml of blood was collected from each naïve rhesus naïve macaque. 9 ml of serum was purified on an immunoaffinity chromatography column as described in Example 5 (System Akta, Pharmacia, Sweden).

Application Buffer: Purification Buffer

Elution buffer: Purification buffer B

The eluates were neutralized with 0.5 M sodium hydrogen phosphate solution. They were then fractionated such that the proteins were present at a high concentration.

μΙ purified against the substance fraction was analyzed on a sorting column (SEC, Zorbax 250 GF). 220 mM phosphate buffer, pH 7 + 10% acetonitrile were used as the partitioning agent (flow rate: 1,000 ml / min, wavelength 214 nm). As can be seen in the chromatogram from this analysis (see Figure 5), the antibody fraction contains IgM (retention time 6.963 min) and IgG (retention time 8.745 min) at a ratio of about 3: 2.

The total amount of antibody fraction was approximately 50 μΙ (determined by SEC compared to the standard, see Figure 6).

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Example 7

Various Purified Antibody Formulations for Vaccine Formulation i

Vaccines were formulated in an ultrafiltration centrifuge unit (Centricon 10, Amicon, USA). The ultrafiltration units were washed by centrifugation with 1 mM phosphate buffer, 0.86% NaCl, pH 6.0 (NBK).

Then 1 ml of buffer (NBK) was filled into the unit and 37 μΙ of 2% Alhydrogel (Superfos Biosector, Denmark) was added. After addition of the neutralized eluate of Example 6, centrifugations and washes (with 5 ml of buffer) were performed according to Centricon instructions.

. The vaccine was then resuspended and made up to 550 μΙ with buffer ä stored in a sterile manner.

Alternatively, the vaccine was made up to 545 μΙ buffer after resuspension and 5.5 μΙ of the timerosal stock solution (10 mg / ml, Sigma, USA) was added.

A third variant of the vaccine formulation was sterile filled into glass vials and thermally denatured in an autoclave (121 ° C, 20 minutes).

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Claims (24)

Use of an antibody for the manufacture of a pharmaceutical composition suitable as a vaccine for therapeutic or prophylactic vaccination against cancer, wherein the antibodies are isolated from body fluids containing antibodies by immunoaffinity purification, wherein antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype are used as ligands for immunoaffinity purification, and wherein body fluids are obtained from an individual that has not previously been administered a monoclonal antibody directed against either of the tumor-associated antigens. The use of claim 1, wherein the pharmaceutical composition is for administration as an autologous individual vaccine to an individual from whose body fluids the antibodies have been obtained. Use of an antibody for the manufacture of a pharmaceutical composition suitable as a vaccine for therapeutic or prophylactic vaccination against cancer, wherein the antibodies are isolated from body fluids containing antibodies by immunoaffinity purification, wherein antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype are used as ligands for immunoaffinity purification, wherein the pharmaceutical composition is for administration as an autologous individual vaccine to a subject from whose body fluids the antibodies have been obtained. P P 0654-2002 31938pn ns / T Use according to any one of claims 1 to 3, wherein the body fluid is serum. Use according to any one of claims 1 to 4, wherein the individual is a human. Use according to any one of claims 1 to 5, wherein several antibodies directed against different tumor-associated antigens and / or different epitopes of such antigens are used simultaneously for immunoaffinity purification. The use of any one of claims 1 to 6, wherein the antibodies used as ligands for immunoaffinity purification are monoclonal antibodies. Use according to any one of claims 1 to 7, wherein the pharmaceutical preparation is prepared repeatedly and is intended for sequential administration for individual vaccination repeated at appropriate intervals. Use according to any one of claims 1 to 8, wherein the tumor-associated antigens are membrane-localized molecules that are often expressed and / or coexpressed on tumor cells of epithelial origin or on tumor cells of neuroendocrine origin or tumor cells of the hematopoietic system. The use of any one of claims 1 to 9, wherein the antibodies purified by immunoaffinity purification or derivatives thereof are formulated together with an adjuvant suitable for a vaccine. PP 0654-2002 31938pn ns / T oO The use of any one of claims 1 to 10, wherein the composition further comprises a protein that enhances the immune response and which is co-administered with the antibodies. The use of claim 11, wherein the protein is a human protein. The use of claim 12, wherein the human protein is a granulocyte-macrophage colony stimulating factor (GM-CSF). Use according to any one of claims 1 to 13, wherein the composition is suitable for administering an antibody purified by immunoaffinity purification as a vaccine by subcutaneous or intramuscular injection. Use according to any one of claims 1 to 14, wherein the antibodies contained in the composition are mixed with an adjuvant and subjected to heat. Use of isolated, ex vivo cultured dendritic cells of an individual for the manufacture of a pharmaceutical composition suitable as a vaccine for therapeutic or prophylactic vaccine against cancer, wherein the dendritic cells have been previously incubated in vitro with antibodies obtained by immunoaffinity purification from body fluids of the same individual containing antibodies wherein the ligands for immunoaffinity purification are antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype, and the body fluids come from an individual who has not previously received a monoclonal antibody directed against either of the tumor-associated antigens. P P 0654-2002 31938pn ns / T 17. A pharmaceutical composition comprising antibodies obtained from body fluids containing antibodies by immunoaffinity purification, wherein for immunoaffinity purification, antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype have been used as ligands, and wherein body fluids come from an individual who has not previously received a monoclonal antibody directed against either of the tumor-associated antigens. 18. A pharmaceutical composition comprising an isolated ex vivo cultured dendritic cell of an individual, wherein the dendritic cells have previously been incubated in vitro with antibodies obtained by immunoaffinity. purification from body fluids of the same individual containing antibodies, wherein antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype were used as ligands for immunoaffinity purification, and body fluids originate from an individual who had not previously been administered a monoclonal antibody or against one of the tumor-associated antigens. 19. A method of making an antibody preparation, wherein the antibodies are isolated from body fluids containing the antibodies by immunoaffinity purification, wherein for immunoaffinity purification, antibodies recognizing one or more tumor-associated antigens or fragments thereof having the same idiotype are used as ligands, and wherein body fluids are derived from an individual who has not previously received a monoclonal antibody directed against either of the tumor-associated antigens. PP 0654-2002 31938pn ns / T Μ Method according to claim 19, characterized in that several antibodies directed against different tumor-associated antigens and / or epitopes of such antigens are used simultaneously for affinity purification. The method of claim 19 or 20, wherein the antibodies used for affinity purification are monoclonal antibodies. The method of any one of claims 19 to 21, wherein the body fluid is serum. A process for the manufacture of a pharmaceutical composition, characterized in that the antibody preparation is prepared by a method according to any one of claims 19 to 22 and the antibody preparation thus obtained is formulated with a pharmaceutically acceptable carrier and / or adjuvant suitable for a vaccine. 24. The method of claim 23, wherein the antibody preparation is admixed with an adjuvant and then subjected to heat. PP 0654-2002 31938pn ns / T 1/6 Fig. 1 2/6 Fig. 2 3/6 OD x 1000 dilution 1: normal human IgG affinity purified antibodies Fig. 3 OD x 1000 4/6 - O O v- O X serum dilution 1: 100000 ^J -s- pre-serum immune serum Fig. 4 5/6. Fig. 5 6/6