EP2015772A2

EP2015772A2 - Agent for the treatment of malignant diseases

Info

Publication number: EP2015772A2
Application number: EP07724827A
Authority: EP
Inventors: Elke Pogge Von Strandmann; Andreas Engert
Original assignee: Individual
Current assignee: Individual
Priority date: 2006-05-03
Filing date: 2007-05-03
Publication date: 2009-01-21
Also published as: WO2007144046A2; WO2007144046A3; US20100291105A1

Abstract

The invention relates to an agent for treating tumor diseases such as multiple myeloma. Said agent acts upon NK cells by activating an anti-tumor immune response after stimulating the NKp30 receptor and the natural cytotoxicity receptor (NCR). Said agent comprises a physiologically effective amount of BAT3 protein, BAT3/anti-CD138 protein, BAT3-specific antibodies, or derivatives of said substances in an acceptable carrier material. The invention can also be used for treating CD138-negative tumors according to the same principle of the BAT3/anti-CD138 protein, wherefore the anti-CD138 component containing an antibody fragment is replaced with any tumor antigen, and the corresponding agent is used for treating tumors expressing said tumor antigen. The invention further relates to the use of recombinant BAT3 protein or a BAT3 fragment without an antibody-based fusion component for treating malignant diseases by activating NKp30 and NCR on NK cells. The invention finally relates to the use of BAT3 cDNA for introducing BAT3 in vivo and/or ex vivo into tumor cells, which results in better recognition by NK cells, in immunotherapy of malignant diseases.

Description

Agent for the treatment of malignant diseases

The invention relates to an agent for the treatment of tumor diseases, autoimmune diseases or for use in cellular immunotherapy in the context of allogeneic and autologous transplants by activation or inhibition of BAT3 with a physiologically effective amount of the protein BAT3 (HLA-associated transcript 3) and / or BAT3 in conjunction with a tumor-specific antibody fragment, the DNA (cDNA) and / or RNA coding for the production of such an agent in the case of activation of the BAT3-dependent disease. Furthermore, the invention relates to the use of BAT3 cDNA for ex vivo and in vivo overexpression of BAT3 in tumor cells after viral or non-viral gene transfer, with the aim of eliciting an anti-tumor immune response in tumor patients. By and large, a means of inhibiting or activating BAT3 by antagonistic or agonistic antibodies, antisense nucleotides or specific inhibitors. Furthermore, a diagnostic marker, such as a specific antibody for the diagnosis of the disease and for the follow-up.

description

The invention relates to an agent for the treatment of human diseases using the recombinant protein BAT3, the coding DNA / RNA, an inhibitor / activator of BAT3 in the form of

Antibodies or antisense nucleotides or other specific inhibitors and using BAT3, which can be obtained via a suitable linker in

Compound with a single-chain antibody fragment of the mouse monoclonal antibody B-B4, which is directed against the tumor-specific antigen CD138, is synthesized.

Furthermore, the invention relates to the use of the BAT3 cDNA for ex vivo and in vivo overexpression of BAT3 in human tumor cells after viral or non-viral gene transfer to achieve an NKp30-mediated immune response against the tumor cells in human malignant diseases.

NK cells, which as a component of the innate immune system recognize malignant cells, combat them directly and contribute to the formation of an adaptive immune response against the tumor cells, have recently been increasingly used for the immunotherapy of malignant diseases.

The activity of NK cells against tumor cells is controlled via activating receptors, which include the natural cytotoxic receptors (NCRs) with the important NKp30. The stimulation of the receptors, as well as that of NKp30, is based on the interaction with the corresponding ligands on malignant cells and leads to the lysis of the target cells (Pende, D., et al., J Exp Med 190, 1505 (Nov 15, 1999)). Thus, the ligand (or possibly the ligands) of NKp30 is basically suitable for tumor therapy. So far, however, the cellular ligand has been described only indirectly with the aid of masking antibodies, and the molecular identification of the ligand was still pending (L. Moretta, A. Moretta, Embo J 23, 255 (Jan. 28, 2004)). Contrary to expectations that a ligand of NKp30 is a membrane protein of tumor cells, we have identified a nuclear protein as a cellular ligand of the NKp30 receptor. In the enclosed manuscript we describe the cloning of BAT3, the first cellular tumor-associated ligand of the surface receptor NKp30, one of the important activating immune receptors of NK cells (Moretta L., A. Moretta, Embo J 23, 255 (Jan 28, 2004) ). Surprisingly, this nuclear protein arrives at the surface of the cells and is also released from the cells as soon as the cells receive a "stress signal." This may be a heat shock or contact with NK cells.

Depending on the context, BAT3 may lead to inhibition or activation of NK cells, which is mainly mediated through the Natural Cytotoxicity Receptors (NCRs).

Thus, BAT3 can lead to activation (cytotoxicity, cytokine secretion) on the cell surface or associated with exosomes in the supernatant of tumor cells. The recombinant, purified protein, on the other hand, inhibits NK cell activity. The addition of BAT3-specific cross-linking antibodies in combination with purified BAT3 in turn leads to a dramatic increase in NK cell activity.

Overall, our data show that the immobilized form of BAT3, whether on the cell surface, on exosomes, or by antibodies, results in NK activation, while the soluble form causes inhibition. This soluble form may arise, for example, by shedding the molecule and thus be a mechanism of tumor and virus-infected cells to escape detection by the immune system. This interpretation is evidenced by evidence of significant increase of BAT3 in serum of tumor patients compared to healthy donors (example Hodgkin's lymphoma comparison of 40 patient sera with 40 normal sera). This is a hitherto unknown or hypothetical mechanism by which important surface receptors of NK cells (NKp30, NCR) are regulated by a nuclear protein (BAT3), whereby the translocation of BAT3 to the cell surface and the cell environment is inducible ,

For all relevant structural data, reference is made to the publications and papers cited therein (NKp30: D. Pende et al., J Exp Med 190, 1505 (Nov 15, 1999), L. Moretta, A. Moretta, Embo J 23, 255 ( Jan 28, 2004) and BAT3: J. Banerji, J. Sands, JL Strominger, T. Spies, Proc Natl Acad. USA 87, 2374 (Mar, 1990)).

It is known that the expression of NKp30-specific ligands on tumor cells correlates directly with their sensitivity to NK cells, so that the loss of ligands represents a strategy of the tumor cells to escape control by the immune system.

However, this not only applies to NKp30, but also to the already known ligands of a second activating receptor of NK cells, the NKG2D receptor. Genherapeutic pre-clinical data are already available, showing that the overexpression of NKG2D ligands leads to the successful control of the corresponding tumor by the immune system. This activation is independent of the activity of the inhibiting NK receptors, which normally prevent NK cells from attacking the body's own cells (eg A. Cerwenka, JL Baron, LL Lanier, Proc Natl Acad. USA 98, 11521 (Sep 25, 2001 A. Diefenbach, ER Jensen, AM Jamieson, DH Raulet, Natūre 413, 165 (Sep 13, 2001)).

NKG2D ligand-based recombinant constructs also show promising anti-tumoral activity, as has been demonstrated in various preclinical studies (Germain, C. et al., Clin Cancer Res 11, 7516 (Oct. 15, 2005); E. Pogge, Strandmann et al., Blood (Oct 6, 2005)). Our identification of BAT3 as a cellular ligand of NKp30 and our studies on the function of BAT3 show that this new ligand BAT3 is suitable for tumor therapy targeting NKp30 / NCR-mediated NK cell stimulation.

Our studies show that overexpression of BAT3 in tumor cells after induction by heat shock or contact with NK cells leads to the release of BAT3 and that activation of NK cells is measurable. Likewise, human tumor cell lines such as 293T and RPMI cells secrete endogenous BAT3, while colon carcinoma lines such as LS175T are unable to do so (see attached manuscript). It can be assumed that BAT3 is functional even without targeting tumor cells via an antibody fragment or via an antibody and can lead to NK cell activation.

The invention therefore also provides an agent for the treatment of patients with malignant diseases, in which a physiologically effective amount of the protein BAT3 is contained as active ingredient in a pharmaceutically acceptable carrier material.

There is a great need for therapies capable of eliciting an anti-tumor immune response from the patient. But also therapies that include transplants or diseases that come from the immunological group are currently far from mature. Thus, in recent years, a variety of immunotherapeutic strategies based primarily on the use of antibodies have been developed or are currently in clinical trials.

An example of the use of BAT3 in tumor therapy is the use as a fusion protein with an antibody fragment that binds specifically to

Allows tumor cells. For example, a BAT3 / anti-CD138 is to be used therapeutically to support an anti-tumor immune response in patients with multiple myeloma, similar to that already described for the bispecific

Protein ULBP2 / CD138 (Pogge et al., 2006). The used B-B4 single chain antibody fragment with specificity for CD138 (Syndecan 1) is basically very well suited for this application. CD138 is a member of Syndecan family, heparan sulfate proteoglycans, play a role in cell adhesion, differentiation and proliferation and are often overexpressed on malignant multiple myeloma cells while lacking on other hematopoietic cells (Dhodapkar MV, Sanderson RD et al., Leuk Lymphoma 1999; 34: 35-43).

In addition, there is still no satisfactory treatment option for the treatment of multiple myeloma. The life expectancy for affected patients after initial diagnosis is only a few months.

Another example is the inhibition of BAT3 in autoimmune diseases to modulate a hyperreactive immune system.

Antagonist and agonistic BAT3-specific antibodies and recombinantly produced BAT3 protein can be used for the therapy of malignant diseases and immunological diseases.

Furthermore, activation of NK cells in the context of cellular immunotherapy by recombinant BAT3 or derivatives / antibodies within the culture phase of these cells before transplantation, as well as systemic administration after transplantation, is a novel approach that improves therapy.

Another example is the diagnosis of diseases and therapy by detecting BAT3 on the cells and in the serum. This can be done by antibody-based techniques, for example ELISA, Fig. 2), but also other specific detection methods.

The invention thus relates to an agent for the treatment of patients with multiple myeloma, in which a physiologically effective amount of the protein BAT3 / antiCD138 or BAT3 and derivatives of BAT3 or BAT3-specific antibodies is contained as active ingredient in a pharmaceutically acceptable carrier material. Other possible uses are malignant diseases and immunological diseases (allergies, autoimmune diseases). The agent according to the invention contains the anti-tumor immune response modulating protein BAT3 and derivatives of BAT3, BAT3 / antiCD138 fusion constructs or BAT3-specific antibodies as active ingredient for the therapy and diagnosis of malignant diseases and for the modulation of the immune system.

The agent of the invention for the treatment of malignant diseases contains the active ingredient in a conventional pharmaceutically acceptable and acceptable carrier material. It may be useful to administer the drug systemically, as it is known for example in the interferon therapy of multiple sclerosis or for the treatment of diabetes.

Further, reference is made to the possibility of ex vivo use in the context of cellular immunotherapies.

It goes without saying that the protein or the DNA and / or RNA coding for it or the specific BAT3 antibodies can be stabilized for storage and for the prevention of premature reduction of activity. Stabilization can be achieved by incorporation of conventional additives, e.g. Buffer substances, salts of other proteins as well as DNA and RNA can be achieved. Examples are albumin, herring sperm, DNA, tRNA and detergents such as Triton, alkali and alkaline earth ions and the like. Storage of the agent and / or active ingredient in dried or freeze-dried form or after

Shock freezing in liquid nitrogen may also be useful.

The agent according to the invention may of course contain the active ingredient in modified form, i. as a protein in which individual amino acids are exchanged or missing. The prerequisite is that the thus-modified active substance still provides the effect it requires in stimulating the immune effector cells for the treatment of patients with malignant diseases. Reasons for such modifications may be the stabilization of the active ingredient, technical or formulation-technical reasons or also an improvement of the effect or the spectrum of action. In question are mutation and fusion after chemical or genetic engineering

Methods and fusions with N- and C-terminal proteins or Peptides. For example, this can lead to an improvement in the half-life of the protein. For reasons of better purification by affinity chromatography, for example, histidine tags or Gst fusions can be made. By phosphorylation or glycosylation on suitable residues modifications can be made, which prevent the degradation by endogenous proteases.

In order to stabilize the protein, it may be useful to make changes, for example at the DNA level, to eliminate, for example, restriction sites, chemical instability or targets for nucleases.

The protein can be obtained in a conventional manner after cloning the gene into suitable vectors recombinant from bacteria or eukaryotes according to standard methods.

The agent according to the invention for the treatment of malignant diseases by the activation of the immune effector cells contains the active ingredient in a pharmaceutically acceptable carrier material, which on the one hand can consist of several conventional constituents. The carrier material expediently contains a transfer medium suitable for the active ingredient. The agent is intended primarily for systemic administration and ex vivo therapy in the context of cellular immunotherapies.

Like a recombinant BAT3 protein or BAT3 fusion protein or BAT3-specific antibodies, the use of the BAT3 cDNA for overexpression of BAT3 in tumor cells by viral and non-viral methods of gene transfer of the invention, since BAT3 overexpimating tumor cells are suitable, via the stimulation of NCR Receptors to support an NK cell-mediated anti-tumor immune response. The invention is further illustrated by the following examples in which experimentally the effectiveness of BAT3 ligand is detected. For a further explanation of the examples and further examples, reference is made to the accompanying manuscript.

5 examples

1. BAT3 overexpression in tumor cells leads to the activation of

NK cells: Increased cytotoxicity against tumor cells

NK cells isolated from the blood of healthy donors were used lo as effector cells in europium release assays. Here are

Target cells (human tumor cell lines) labeled with europium and incubated with NK cells in the indicated EffektorTarget ratios. Subsequently, the amount of europium released is determined in the supernatant, which is used as a measure of the

NK-mediated cell lysis is used. The value for a 100% lysis results after incubation of the cells with a detergent which leads to lysis. In addition, the spontaneous lysis is taken into account, which is determined in batches without NK cells.

(A) The NK cells (effector) were incubated for the europium release assay in the ratios indicated with the human colon carcinoma line LS175T, which with an expression construct for BAT3 (BAT3) or the

20 comparison with the expression plasmid (vector) was transfected. There is an increased lysis of BAT3-expressing cells at all measuring points, which is mediated via NKp30, since a recombinant NKp30 receptor inhibits this effect (BAT3 + NKp30-Ig).

(B) The NK cells (effector) were incubated for the europium release assay in the ratio 5: 1 with the human renal carcinoma line 293T, which was transfected with an expression construct for BAT3 (BAT3) or for comparison with the expression plasmid (vector). There is an increased lysis of the BAT3-expressing cells, which is mediated via NKp30, since the effect by adding a masking NKp30 antibody

30 is inhibited (BAT3 + α-NKp30).

(see FIG. 1) 2. BAT3 overexpression in tumor cells leads to activation of NK cells: Stimulation of Inteferonγ secretion

Expression vectors for BAT3 (BAT3) or for a membrane-bound BAT3 5 derivative (CD3ζ-BAT3-CT) or control vectors without BAT3 cDNA were introduced into LS175T cells. After 36 hours, the tumor cells were mixed with primary NK cells from peripheral blood of healthy donors (see above) in the ratio 1: 1 and incubated for a further 48 hours. The NK cells were previously stimulated overnight with interleukin 2 (10U) alone or in combination with interleukin 15 (10 ng ml ^-1 ) Interferonγ in the supernatants were assayed for interferon g secretion of the NK cells using a IFNγELISA kits (human interferon γ ELISA Kit (R & D Systems)) .The overexpression of BAT3 (upper part of the figure), which is released from the tumor cells into the supernatant (detected in the i5 Western blot, see attached manuscript) and also that of a membranous variant (lower part of the figure) stimulates the interferon g secretion of NK cells (see Figure 2).

20

3. BAT3 is for the detection and elimination of tumors (multiple

Myeloma) essential: in vivo experiment (mouse xenograft model)

Human multiple myeloma cells (line RPMI8226) were injected into nude mice. This leads to the formation of subcutaneous tumors in 8 out of 10 mice. The tumor volume on day 13 and day 20 is indicated (circles). If the mice additionally receive human peripheral blood lymphocytes (PBL), the tumor cells are eliminated and tumor formation does not occur in any case. In addition, the mice were given control antibodies (control antibodies) that do not interfere with tumor cell recognition (black cross). However, when the mice receive BAT3-specific antibodies that deplete the endogenous BAT3 protein, tumor cell recognition and elimination are inhibited (yellow triangles, tumors in 6 out of 10 mice).

This experiment shows that BAT3 is necessary for the recognition and elimination of tumor cells in vivo. (see FIG. 3)

Claims

claims

A composition for the treatment and / or diagnosis of tumor diseases, for example multiple myeloma, by the activation of an anti-tumor immune response after stimulation of the NKp30 receptor and the Natural Cytotoxicity Receptors, characterized in that it is a physiologically effective amount of the Protein BAT3 and / or BAT3 / antiCD138 and / or a BAT3 fragment and / or BAT3-specific antibodies in an acceptable carrier material.

2. Composition according to claim 1, characterized in that the

Carrier material containing the drug-transporting transfer medium.

3. Composition according to one of claims 1 and 2, characterized in that it is present as an injection formulation and is used.

4. Composition according to one of claims 1 and 2, characterized in that it is present as a formulation for ex-vivo use in the context of cellular immunotherapy in tumor patients and is used.

5. Composition according to one of the preceding claims, characterized in that it is used for the therapy of tumors expressing CD138, such as colon, lung or prostate carcinoma.

20 6. Composition according to one of the preceding claims, characterized in that it after exchange of the antiCD138 portion with a Antibody fragment against any tumor antigen, is used for the therapy of tumors expressing this antigen.

7. Use of the protein BAT3 as an active ingredient for the preparation of an agent according to any one of the preceding claims for the treatment of malignant diseases.

8. Use of the protein BAT3 / antiCD138 as an active ingredient for the preparation of an agent according to any one of the preceding claims for the treatment of malignant diseases.

9. Use of BAT3-specific antibodies or antibody fragments as active ingredient for the preparation of an agent according to any one of the preceding claims for the treatment of malignant diseases.

10. Use of the BAT3 DNA and / or RNA for the production of BAT3 / antiCD138 analog constructs, in which the CD138 binding portion is fused with an antibody fragment against any tumor antigen, as an active ingredient for the preparation of an agent according to any one of the preceding claims for the treatment of malignant Diseases that express the tumor antigen in question.

11. Use of the BAT3 cDNA for in vivo or ex vivo overexpression of BAT3 in tumor cells according to the prior art viral or non-viral gene transfer for the immunotherapy of malignant diseases.