WO2001080904A2

WO2001080904A2 - Means for diagnosing and treating carcinomas

Info

Publication number: WO2001080904A2
Application number: PCT/DE2001/001530
Authority: WO
Inventors: Stefan Dübel; Andreas Schmiedl
Original assignee: Fischer, Peter; SCHERBERICH, Jürgen, E.
Priority date: 2000-04-22
Filing date: 2001-04-23
Publication date: 2001-11-01
Also published as: WO2001080904A3; DE10020034A1; AU2001265749A1

Abstract

The invention relates to means for diagnosing and treating carcinomas, in particular, kidney cell carcinomas, based on recombinant substances which are binding partners of gamma-glutamyltransferase (=gamma-glutamyltranspeptidase, GGT, EC 2.3.2.2.). Said substances can be used in the medical field and in the pharmaceutical industry. The aim of the invention is to develop reliable methods for diagnosing and treating kidney cell carcinomas, based on methods which currently exist that use native antibodies such as mAK 138H11. The invention is characterized in that it uses a manufactured recombinant ligand against gamma-glutamyltransferase, in particular a recombinant antibody or antibody fragment. A preferred recombinant antibody is illustrated in figure 2.

Description

Means for diagnosis and therapy of carcinomas

The invention relates to an agent for the diagnosis and therapy of carcinomas, in particular renal cell carcinomas, based on recombinant substances which are binding partners of gamma-glutamyltransferase (= gamma-glutamyltranspeptidase, GGT, EC 2.3.2.2.). Areas of application are medicine and the pharmaceutical industry.

Renal cell carcinoma

In the United States alone, over 30,000 new cases and 12,000 deaths from renal cell carcinoma (RCC) were recorded annually for 1996 to 1998. The Munich Tumor Registry (www.krebsinfo.de) registered 6612 deaths in 1995 by the RCC for Germany. Mortality of over 100,000 is estimated worldwide by the RCC for the year 2000, the incidence is increasing dramatically [1].

Men get sick more often than women, and the disease usually manifests itself in the fifth to sixth decade of life. RCC is rarely observed in children, but it is difficult to differentiate it from other kidney tumors such as Wilm's tumor from a differential diagnosis. RCC has recently been observed in children as a late consequence of neuroblastoma chemotherapy. The prognosis of progressive kidney cancer is equally unfavorable in children and adults with a median survival time of less than 26 months (Munich tumor registry). In stage III the five-year survival rate is 15 to 35%, in stage IN only 0 - 10%. According to international literature, distant metastases are found in about 30% of patients even when the primary tumor is diagnosed; in a further 40% of the patients, late metastases develop after primary tumor resection. Due to the earlier diagnosis (ultrasound), this percentage is decreasing, but with a drastically increasing incidence.

To date, there is no effective systemic therapy for patients with metastatic RCC. Chemotherapy (cytostatics) or radiation treatment are practically ineffective, and therapy attempts with interleukin-2 and / or interferons have also been found if the numerous published studies are summarized, so far overall less than 25% objective tumor regressions (CR + PR) combined with starch side effects [ 1,2].

Immunotherapy with the mouse antibody 138H11

The monoclonal antibody (138H11) against human, renal gamma-glutamyl transferase (GGT, EC 2.3.2.2)) was produced by Fischer & Scherberich [3,4]. The mAb 138H11 reacts specifically with GGT human tissue on a large number of frozen sections. The strong reaction with almost all investigated clear cell cells was remarkable and chromophilic (= papillary) kidney carcinomas, which together make up over 85% of all malignant kidney cell carcinomas. Immunohistochemically, the mAb 138H11 thus enabled a differential diagnostic classification (according to Störkel and Thoenes) of epithelial kidney tumors in two groups: one over 98% positive (clear cell and chromophilic kidney Ca) and one 100% negative (oncocytomas, chromophobic and duct Bellini Ca) [2,3,5,6].

The mAb 138H11 also reacted with proximal tubular cells of the kidney. The polarization of the GGT distribution was striking: almost exclusively the luminal (apical) part of the cells (brush border) contained immunoreactive GGT. An extremely weak but specific marking in the basal cell pole was only visible in individual kidney sections. In contrast to normal tubular cells, the tumor cells of the clear cell renal carcinomas no longer showed polarization; the entire cell membrane and z. T. also the cytoplasm reacted with mAb 138H11 [5]. In the liver, mAb 138H11 only reacted with the GGT in the biliary tubules. A positive reaction was also found in gliomas [7], hepatocellular carcinomas and in gastric carcinomas as well as in RCC liver metastases [2,3].

Immunoscintigraphy with ^99m Tc-labeled mAb 138H11

Tumor-bearing human kidneys were perfused extracorporeally after the nephrectomy. There was a significant uptake of the radioactively labeled antibody in clear cell renal carcinoma. This was up to 20 times higher in the tumor tissue than in the renal cortex [8,9]. In contrast, no antibody was enriched in a GGT-negative oncocytoma. This high accumulation in the tumor can be explained by the fact that in normal kidney cells the target antigen is expressed almost exclusively in apical membranes (brush border of the proximal tubule). In such polarized cells, the antigen is therefore not accessible via the blood circulation or for the perfusion solution, and only a small amount of mAb was bound to normal kidney tissue during the perfusion. In renal carcinoma cells, the polarization of the cells is obviously disturbed and the antigen is freely accessible to the circulating antibody on the outer membrane. The monoclonal antibody 138H11 therefore recognizes the target antigen not because of increased antigen expression in the tumor, but because of the changed target antigen localization on tumor cells [8] (FIG. 1) [2,10].

138H11-Drugkonjugate

To increase the potential cytotoxicity of the mAb 138H11, we constructed drug conjugates with a synthetic calicheamicin developed at the Scripps Research Institute in La Jolla. Calicheamicin (M _r 1500, from the class of Enediyne antibiotics) leads to double-strand breaks in the DNA. Calicheamicin-Θ is a biological highly active and selective Enediyne molecule, which has been designed in such a way that, in contrast to natural calicheamicin, it is biochemically stable and is therefore very suitable for coupling reactions with mAbs. In the first in vitro investigations on RCC cell lines we were able to show that calicheamicin-Θ has a cytotoxic effect in concentrations that are up to five orders of magnitude lower than known cytostatics. By coupling to the mAb 138H11, this efficiency was only slightly reduced or even increased depending on the linker; the conjugate was therefore extraordinarily cytotoxic [11]. In in vivo experiments with nude mice, the growth of RCC xenografts with these drug conjugates was successfully and specifically inhibited (compared to a control antibody) [12]. In the animal model, the principle effectiveness of antibody therapy against renal cell carcinoma with GGT as target antigen was shown.

Purpose of the invention:

However, the substances successful in animal experiments are only poorly suited for the therapy of humans. Previous attempts at therapy of tumors with monoclonal antibodies from the mouse, such as the 138H11 directed against GGT, have led to severe side effects for patients, in particular by eliciting an antibody immune response against the therapeutic agent (HAMA response). The HAMA antibodies formed in this way prevent multiple use of the therapeutic antibody, since the patient quickly neutralizes further doses through the HAMA antibodies that are then formed. In addition, severe allergic reactions are possible. There are also limits to the therapeutic effect of the native mouse antibody, since the murine effector part, the Fc fragment, is only insufficiently bound by the human effector cells. Mouse IgGl antibodies such as 138H11 are particularly problematic because they do not indicate complement activation, an important component of tumor cell destruction. Although the therapeutic efficiency of mouse antibodies can be significantly increased by chemically coupling toxins, cytostatics or radionuclides as in the known chemoimmunoconjugate with calicheamicin theta, the disadvantages described above remain. The stability and quality control of such chemical couplings is also very problematic.

The available results were therefore not suitable for establishing reliable methods for the diagnosis and therapy of renal cell carcinomas. The object of the invention was therefore to develop such methods. The invention is implemented according to the claims, the subclaims are preferred variants.

An essential part of the invention is the use of a recombinantly produced ligand, in particular a recombinant antibody or antibody fragment. A particularly preferred recombinant antibody is shown in FIG. 2.

The recombinant therapeutic agent against gamma-glutamyltransferase produced according to the invention therefore enables in particular:

1. Avoiding a HAMA response against the therapeutic agent, and thus fewer side effects for the patient

2. Repeated use of the therapy due to a lower immune response against this agent

3. The efficient production of the agent in the form of a fusion protein, thus a defined stoichiometry and defined coupling points

4. Possibility of reduced mass production by recombinant expression in E. coli or other mass cultures (Pichia Pastoris, Saccharomyces, CHO cell culture), or in transgenic plants or animals.

Furthermore, the invention enables the construction of the constructs exemplified in the examples, such as chimeric antibodies, fusion proteins, bispecific antibodies, immunocytokines etc., from the DNA of the antigen binding site of the mAb 138H11. Whose DNA or amino acid sequence also enables direct humanization of the GGT binding partner z. B. by CDR crafting or the like. Recombinant fragments can also bring significant advantages such as faster blood clearing and better tumor penetration for radioimmuno diagnostics and therapy, since their size can be varied depending on the application, e.g. for rapid excretion by the kidney, which is not possible with complete IgG molecules.

The following exemplary embodiments explain the invention without restricting it to these examples. Examples:

Example 1:

The agent can be used to treat tumors (especially renal cell carcinoma) by using the recombinant antibody fragment as a fusion protein, e.g. B. with Interleukin 2 (IL2) is produced (Fig. 3). Systemic administration of IL2 is one of the few treatments for renal cell carcinoma that have been shown to have an impact on the tumor. The use of the fusion protein instead of native IL2 increases the local concentration of IL2 on the tumor and therefore leads locally to an increase in the immunostimulating effect compared to the systemic administration of IL2. This can significantly reduce systemic toxicity.

Production: The two gene fragments for the variable regions (Vh and VI) of the antibody against GGT are obtained via a polymerase chain reaction with oligonucleotide primers which bind specifically to the antigen-binding (V-) regions of antibodies. The amplified genes of the two V regions are provided with the interfaces for further cloning by this PCR and then joined together in the form of an scFv antibody fragment, ie their open reading frames are linked without a stop codon by a DNA fragment which is a peptide encoded by 15-18 amino acid residues. The corresponding scFv gene fragment is then also linked to the gene for IL2 in the same reading frame. At the end of the human IL2 gene, also amplified by PCR, a stop codon is encoded, followed directly by the recognition sequence for Xbal. This gene cassette is cloned into the bacterial expression vector pOPElOl via the restriction sites Ncol and Xbal. After transfection of the construct produced in this way in E. coli, the promoter of the pOPElOl vector is switched on by administration of IPTG for three hours, during which the cells are to be shaken at 28 ° C. The scFv-IL2 fusion protein accumulates in the periplasm of the E. co / z ^' cells during this time. After the end of the production time, the cells are harvested by centrifugation at 5000 xg for 10 minutes and transferred to a buffer which enables the outer membrane of the E. coli to dissolve (50 mM Tris (hydroxymethyl) aminomethane / ΗCl pH 8.0 with 20% w / v) sucrose and 1 mM EDTA), and incubated for 20 min at 0 ° C with shaking. The supernatant obtained after centrifugation at 30,000 xg for 30 min contains approx. 50-1000 μg / ml of the scFv-IL2 fusion proteins, which are further purified, characterized and prepared for therapeutic use via a sequence of affinity chromatography and ion exchange chromatography according to the prior art can. Example 2:

The agent can be used for the therapy of tumors (in particular kidney cell carcinomas) by coupling the recombinant antibody fragment with an enzyme which converts a systemically applied, less toxic precursor substance into a tumor-damaging substance ("ADEPT"). Thus, after administration of a molecular complex consisting of the recombinant antibody against gamma-glutamyl transferase and carboxypeptidase G2 (CPG2), CPG2 can be accumulated in the tumor. After that, the patient is given time to excrete excess molecular complexes before the precursor substance (the "prodrug", in the case of CPG2 these are benzoic acid mustard-derived prodrugs) is given. (see Fig. 4). Lactamases, pH changes in the tumor environment, intracellular reductases or catalytic antibodies can also be used to generate the tumor-damaging substance from the prodrug.

Example 3:

The agent can be used to treat tumors (especially renal cell carcinomas) by presenting the recombinant antibody fragment on the surface of T lymphocytes. This can be done by genetic fusion to the zeta chain of the T cell receptor of the T lymphocyte (FIG. 5), or by loading modified T lymphocytes with recombinant antibody fragments via specially introduced surface molecules of the T lymphocytes. The latter surface molecules can be anchor molecules expressed by the T-lymphocytes, in particular anti-peptide "tag" or anti-hapten antibodies, the recombinant antibody fragment against gamma-glutamyl transferase with the corresponding peptide "tag" or the corresponding one Hapten was modified (Fig. 6). It is also possible to load the T lymphocytes by treatment with agents which anchor the recombinant antibody fragment against gamma-glutamyl transferase in the surface of the T lymphocytes. This is possible by producing fusion proteins from the recombinant antibody fragment against gamma-glutamyl transferase with a surface protein of the Newcastle Disease Virus (in particular hemagglutinin). These viruses fuse with the membrane of the target cell and integrate their hemagluttinin into the target cell membrane, and thus also the recombinant antibody fragment against gamma-glutamyl transferase. This procedure has the advantage that no transformation of the T lymphocytes is necessary. Another possibility of anchoring the recombinant antibody fragments against gamma-glutamyl transferase in the surface of T-lymphocytes is the production of bispecific antibodies (FIG. 7). With one binding arm they bind gamma glutamyl transferase, with the other an epitope on the T lymphocytes. The latter binding to the T lymphocytes can be mediated either by an antibody fragment or by a natural ligand of the epitope. This epitope in turn on the T- Lymphocytes can either be contained in a natural surface molecule of the T lymphocytes (in particular CD4, CD5, TCR, CD28), or it can have got onto the surface of the T lymphocytes by fusion of virus particles, in particular NDV viruses. Several bispecific constructs can be used together to generate the primary and secondary activation signal for the T lymphocyte. The use of bispecific antibodies also has the advantage that no transformation of the T lymphocytes is necessary.

All of the methods listed in this example can also be used for other effector cells, in particular for natural killer cells.

Example 4:

Gene therapy of GGT-positive carcinomas with vectors such as immuno-liposomes or viruses or DNA, which targeting either have a negative effect on the tumor cell (toxicity, apoptosis, reduction in division) or a component that activates the immune system (eg B-7, IL- 2) express. The recombinant antibody is integrated into the liposomes in a known, chemical way or incorporated into the genome of the viruses (e.g. retroviruses, adenoviruses) from a molecular biological point of view. For a DNA or RNA vaccine, the coding sequences of the antibody are integrated into a suitable expression plasmid.

Example 5:

Acidification with anti-idiotypic antibodies or their antigenic components, also as DNA or RNA (see Example 4), which are obtained against GGT binding partners in vitro, in vivo, from antibody libraries, transgenic animals etc. or when applied to humans. This leads to an activation of the tumor patient's immune system, which is directed against the tumor and can thereby control or destroy it.

Example 6:

Isolation of tumor cells from body fluids (bone marrow, blood, urine, cerebrospinal fluid) via GGT binding partners for therapy (e.g. purging for bone marrow or stem cell transplantation, for in vttro gene transfer) or diagnostics (detection of "minimal residual disease" (MRD), relapse etc.) For this purpose, the binding partner of the GGT can be coupled, for example, to magnetic beads, with which a tumor can be used to enrich individual tumor cells from a large excess of irrelevant cells.

Claims

claims:

1. Means for the diagnosis and therapy of carcinomas, in particular renal cell carcinomas, on the basis of substances which are binding partners of gamma-glutamyl transferase (GGT, EC 2.3.2.2) or their isoforms or enzymatically inactive variants.

2. Composition according to claim 1, characterized in that the binding is carried out by a natural or synthetic ligand of gamma-glutamyl transferase or a modification thereof.

3. Composition according to claim 1 and 2, characterized in that the binding to gamma-glutamyl transferase is ensured by binding partners whose binding region on the gamma-glutamyl transferase overlaps with one of the binding partners, in particular with the epitope of the monoclonal antibody 138H11.

4. Composition according to claim 1 to 3, characterized in that the binding to gamma-glutamyl transferase is carried out by a recombinantly produced ligand, in particular by a recombinant Antiköφer or by a recombinant Antiköφerfragment.

5. Composition according to claim 1 and 4, characterized in that the recombinant Antiköφer or the recombinant Antiköφerfragment, which causes the binding to gamma-glutamyltransferase, is derived from a hybridoma-Antiköφer (monoclonal Antiköφer), in particular from the monoclonal Antiköφer 138H11, sequence of the antigen-binding regions s. Fig. 2).

6. Composition according to claim 1 to 5, characterized in that the recombinant Antiköφerfragment contains amino acid sequences of the variable regions of FIG. 2.

7. Composition according to claim 6, characterized in that the binding region of a recombinant or synthetic ligand of the GGT, in particular an antibody fragment which is at least 80% homologous to one of the hypervariable regions (= CDR), in particular the CDR3 of the heavy or light chain ( see Fig. 2).

8. Composition according to claims 1-7, characterized in that the binding partners of the gamma glutamyl transferase by selection from a molecular library, in particular with Help from phage display or ribosome display or modifications of these methods were obtained.

9. Composition according to claims 1-8, characterized in that the binding partners of the gamma-glutamyl transferase in transgenic animals, in particular mice, or in in vitro systems, are obtained after GGT, epitopes or mimotopes of GGT or preparations of tissues or cells containing GGT, epitopes or mimotopes of GGT were used for immunization.

10. Composition according to claims 1-9, characterized in that the binding partners of the gamma glutamyl transferase by humanization, chain shuffling, affinity maturation, mutations to change the specificity or to improve the production, by shortening, or by other modifications of the primary sequence from the monoclonal antibody 138H11 were derived.

11. A composition according to claim 1 based on substances which are binding partners of the gamma-glutamyl transferase coupled to other substances.

12. Composition according to claim 11, characterized in that the binding partner is coupled to substances which can damage or kill the cells in the vicinity of the gamma-glutamyl transferase, in particular to toxins or RNases, or to molecules which form the formation of toxins from precursors effect, or at such preliminary stages.

13. A composition according to claim 11, characterized in that the binding partner is coupled to substances which emit radioactive radiation, in particular to substances whose radioactivity serves therapeutic or diagnostic purposes.

14. Composition according to claim 11, characterized in that the binding partner is coupled to other substances which cause destruction of cells in the vicinity of the gamma-glutamyl transferase by the body's own effectors, in particular by other cells, especially the immune system, such as T-lymphocytes , NK cells, granulocytes or macrophages, or by molecules of the immune system, such as complement proteins.

15. Composition according to claim 11, characterized in that the binding partner of the gamma-glutamyl transferase is anchored on the surface of an effector cell, in particular by coupling to components of the T cell receptor, or by one on the Surface of the effector cell presented specific binding molecule for the molecule that contains binding partners of the gamma glutamyl transferase.

16. Composition according to claim 11, characterized in that an agent which contains the binding partner of the gamma-glutamyl transferase is produced recombinantly as a fusion protein, in particular with cytokines such as interleukins or chemokines.

17. Composition according to claim 11, characterized in that an agent which contains the binding partner of the gamma-glutamyl transferase is prepared by adding anchor domains which allow coupling to an effector, in particular avidin, streptavidin or mutations of these molecules, or biotin , or streptavidin / avidin binding peptides, or bacterial immunoglobulin binding molecules such as protein A, protein G, protein H, protein L, or calmodulin, or calmodulin binding molecules, or fragments of RNases, or unnatural sequences, which bind to fragments of RNases , or leucine zipper.

18. Agent according to claim 11, characterized in that an agent which contains the binding partner of the gamma-glutamyl transferase is coupled to one or more polyethylene glycol molecules.

19. Use of the agent according to claims 1-18, characterized in that it is used for the diagnosis or therapy of tumors which express gamma-glutamyl transferase, in particular kidney carcinomas, gliomas, liver carcinomas, gastric carcinomas, ovarian carcinomas, melanomas or breast carcinomas.

20. Use of the agent according to claims 1-18, characterized in that it is used for the diagnosis or therapy of autoimmune diseases, in particular of rheumatoid arthritis.

21. Use of the agent according to claims 1-18, characterized in that it is used for the diagnosis or therapy of metabolic disorders, in particular disorders of leukotriene synthesis, formation of mercapto acids or glutathione.

22. Use of the agent according to claims 1-18, characterized in that it is used for the (differential) diagnosis of tumor diseases, in particular in immunoscintigraphy, immunohistochemistry or in immunoassays such as ELISA or for the detection of MRD, metastases or relapse to differentiate Gamma- Glutamyltransferase-positive tumors such as kidney tumors, gliomas or adenocarcinomas of unknown origin (CUP syndrome).

Related publications

1. Fischer P, Schmidt C, Kaufmann O, Dietel M, Baum RP, Wrasidlo W, Scherberich JE, Gaedicke G. Experimental immunoscintigraphy and differential diagnosis with the monoclonal antibody 138H11. In: Schnorr D, Loening SA, (eds.) Renal cell carcinoma - Renal Cell Carcinoma. Berlin, Vienna: Blackwell Wiss.-Verl .; 1998: 20-28.

2. Fischer P, Baum RP, Tauber M, Boeckmann W, Störkel S, Weier S, Scherberich JE. Novel monoclonal antibody 138H11 against human gamma-glutamyltransferase: classification, histogenesis and immunoscintigraphy of renal tumors. In: Staehler G, Pomer S, (eds.) Basic and clinical research on renal cell carcinoma. Berlin, Heidelberg: Springer; 1992: 148-155.

3. Fischer P, Scherberich JE, Schoeppe W. Comparative biochemical and immunological studies on gamma-glutamyl transferases from human kidney and renal cell carcinoma applying monoclonal antibodies. Clin.Chim.Acta. 1990; 191: 185-200.

4. Fischer P, Scherberich JE. A monoclonal antibody to gamma-glutamyl transferases of human kidneys and renal tumors. Biol.Chem.Hoppe Seyler. 1989; 370: 620 (abstract)

5. Fischer P, Störkel S, Haase W, Scherberich JE. Differential diagnosis of histogenetically distinct human epithelial renal tumors with a monoclonal antibody against gamma-glutamyl transferase. Cancer Immunol.Immunother. 1991; 33: 382-388.

6. Fischer P, Scherberich JE, Schoeppe W. Monoclonal antibodies to gamma-glutamyl transferase and associated glycoproteins of human kidneys and renal cell carcinomas. In: Streilein JW, et al., (Eds.) Advances in gene technology: the molecular biology of immune diseases and the immune response. ICSU Short reports 10: Proc. Miami Bio / Technology Winter Symp .; 1990: 50

7. Schäfer C, Bahn H, Giese A, Fischer P, Holzhausen H-J, Fels C, Wellman M, Nisvikis A, Rainov ΝG. Gamma-glutamyltransferase expression in malignant gliomas in culture and in vivo. Proc.Am.Assoc.Cancer Res. 1999; 40: 674 (abstract)

8. Fischer P, Baum RP, Tauber M, Boeckmann W, Weier S, Scherberich JE. Immunoscintigraphic localization of renal tumors in an extracoφoreal perfusion model with a monoclonal antibody against gamma-glutamyltransferase. Cancer Immunol.Immunother. 1992; 35: 283-288.

9. Rachel U, Baum RP, Fischer P, Jonas D, Scherberich JE. Monoclonal antibody 138H11 in immunoscintigraphy of human kidney tumors ~ in vitro results. Investig. Urol.Berl. 1994; 5: 66-68.

10. Fischer P, Scherberich JE. Hybridomas reveal shared immunodominant epitopes of gamma-glutamyltransferase isoforms from human kidney and renal cell carcinoma. Tumor Biol. 1996; 17: 369-377.

11. Schmidt CS, Wrasidlo W, Kaufmann O, Scherberich JE, Gaedicke G, Fischer P. Monoclonal antibody 138H11 against gamma glutamyltransferase provides a possible tool for targeting calicheamicin to renal cell carcinomas. Adv.Exp.Med.Biol. 1998; 451: 431-436.

12. Fischer P, Knoll K, Scherberich JE, Gaedicke G, Wrasidlo W. Targeted therapy of renal cell carcinoma xerografts with a chemoimmunoconjugate of mAb 138H11 and calicheamicin θ. Proc.Am.Assoc.Cancer Res. 2000; 41:79 (abstract)