DE10350131A1 - New fusion protein, useful for treating leukemia and solid tumors, comprises specific antigen-binding, microtubulin-binding and immune response-inducing regions, also related nucleic acid - Google Patents

Abstract

The invention relates to the fields of tumor physiology and biotechnology.
The object of the invention is to modify penetrating antibodies, preferably IgG, in such a way that they inhibit cell growth and thus can damage tumor and protozoan cells.
The object of the invention is achieved by fusion proteins containing antibody binding and microtubule binding regions.

Description

The The invention relates to the fields of tumor physiology and biotechnology.

In of tumor therapy provide surgery, radiation and chemo as before, the decisive measures for the treatment of the disease In chemical tumor therapy (chemotherapy) are ever according to tumor type usually cytostatic agents of different modes of action used, so such as alkylating agents, nitrosourea compounds, folic acid antagonists, Pyrimidine and purine analogues such as fluorouracil, antibiotics with effect on the DNA-dependent RNA polymerase or enzymes such as L-asparaginase. A group of cytostatics for chemotherapy are the mitotic inhibitors such as taxol and vinca alkaloids.

On The reason for their very good anti-tumor activity is especially the mitosis inhibitors reinforced recently Attention found. The mitosis inhibitors influence the construction or Degradation of microtubule dividing spindle - and thus attack at the cell division. The known colchicine or vinca alkaloids bind to specific Binding sites of the α- or β-tubulin - as a building block the Miktotubuli - and cause, e.g. an inhibition of the structure of the microtubules. Other Mitiosegifte - for example the taxol - causes their destabilization.

mitosis or spindle toxins are highly toxic and are therefore therapeutic Purposes problematic. The toxicity of colchicine is even so high that these Substance has not been used therapeutically so far. The end Yew (Taxus) isolated alkaloid Taxol is currently the subject of intensive Research.

The Most mitotic inhibitors bind to the microtubule β-tubulin. To they have binding sites whose different high specificity for a classification the mitosis inhibitor is used. So are different groups such as the colchicine type, the taxane type, the vinca alkaloid type or the rhyoxin type distinguished.

On Reason of high toxicity The cytostatics is a therapy with these substances with many side effects connected to that for the affected patients are often hardly bearable. Therefore, for many Years of improving therapies with the objective of Avoidance or reduction of side effects worked. An approach this is the attempt to target the active ingredients only to the treating cells - d. H. to the target cells - too to steer.

A possibility to implement this approach is essentially based on identify cell-type specific epitopes, an epitope-specific monoclonal antibody to produce and the thus obtained antibody or antigen-binding To couple fragments thereof with a therapeutically active molecule. Such an approach is the subject of a research project of the university from California with the aim of a specific therapy of breast cancer (Sherie L. Morrison, Ph.D .: "Antibody Fusion Proteins for the Therapy of Breast Cancer ", University of California, Los Angeles, 1997-1999). This was antibodies used against the breast cancer specific molecules her2 / neu and CEA and associated with immunostimulatory molecules that increase the activity of T cells stimulate.

Even though this approach is associated with the benefit of high therapeutic selectivity, he is in practice only with great effort at high To implement effort and long development time, since numerous development steps necessary for its realization. For this you must first for each Cell-type specific antigens are isolated. As it is with these usually protein antigens, will be cell-specific in the following Epitopes of the antigen determines the lowest possible similarities to epitopes of the proteins of other cell types. This is required to avoid cross-reactivity of the therapeutically used Antibody. Subsequently the production of monoclonal, against the respective antigen directed antibody, in the further elaborate selection and / or mutagenesis Such as phage display must be subjected to an antibody as possible high specificity, or as possible low cross-reactivity too reach.

In addition, difficulties often arise in the preparation of the ready-to-use therapeutic agent, since a non-human antibody must be modified in order to be able to be used without high allergenic potential. For this purpose, the variable regions, but in particular the complementarity determining regions (CDR) can be used in a human antibody scaffold, in the finished therapeutic different sized antigen-specific elements of the therapeutic antibody, such as the antigen-binding fragments (Fab) are used. These are usually antigen-specific elements that consist of at least two separate polypeptide chains. The preparation of these complex antigen-specific elements and their linkage with the actually therapeutic molecule is often expensive in practice and requires complex expression constructs and correspondingly suitable host cells. Disadvantages are that penetrating antibodies are either not modified at all or with radioactive isotopes or with cholera toxins.

The The object of the invention is penetrating antibodies, preferably To modify IgG to inhibit cell growth and thus Damage tumor and protozoan cells can.

The The object of the invention is fusion proteins containing antibody binding and Microtubule binding regions dissolved.

The The effect of these fusion protein-antibody complexes is to Mikrotubuli- or cytoskeleton to find or bind after the the tumor cells bind their target cells by high-affinity antibodies and penetrate.

The antibody Binding region is e.g. Staphylococcal protein A (SPA), extracellular region Fc receptor CD 64 etc.

The Microtubule binding region is e.g. Gephyrin, MID-1, MAP, Tau etc.

These Fusion proteins can also long and super long spacer regions such as e.g. polyglycine or Polyproline Containing Membrane Penetration Domain (MBD) or protein transduction domain (PTD) are merged.

The Fusion proteins can from immune-triggering Regions such. B. Fc, B 7.1 or B 7.2 contain the effect to increase the complexes.

The Fusion proteins can Nucleic acid or Polysaccharide-binding domains or with which they are merged in order to increased Concentration to enhance the effect.

The Fusion proteins can be fused with GFP or other fluorescent proteins to to visually pursue their effects or their concentration intensity to measure the fluorescence.

Besides, these can Fusion proteins hinge regions, such as e.g. Five-glycine regions and at least one GST, Histag or another region to perform the affinity purification contain.

The The protein transduction domain (PTD) described is one eleven amino acid long Region representing a region of the HIV Tat protein.

the Researcher Dowdy and his colleagues managed to get 60 proteins in the sizes order between 15 kDa and 120 kDa and following denaturation transport the fusion with urea into the cytosol. (Science 285, 1569-1572, 1999 and Nature biotechnology Vol. 17 p. 942, Oct. 1999).

To the internalization of the fusion protein, the microtubule-binding domain acts as e.g. Gephyrin, Tau, MAP or MID-1 in the cytosol. It binds microtubules and thus captures the cytoskeleton. The dynamic balance (Wilde et al., Nature Cell Biology 2001, March, vol. 3 and Carazo. Salas et al. Nature Cell biology 2001, March, vol. 3.) of the microtubules impaired and the cell can not divide anymore. As soon as you she does not share anymore, she dies.

Thereby the growth of the tumor, e.g. a solid tumor inhibited.

In the 1 the IgG antibody-SPA-MBD fusion protein complex is shown schematically.

In the 2 the IgG antibody-fusion protein complex is shown schematically. The fusion protein contains PTD, long spacer, SPA and MBD.

In the 3 the IgG antibody-fusion protein-DNA complex is shown schematically.

The Contains fusion protein SPA, MBD and a DNA binding domain, the DNA binds.

In the 4 the IgG antibody-SPA-MBD-GFP fusion protein complex is shown schematically.

In the 5 the IgG antibody-fusion protein complex is shown schematically.

The Contains fusion protein SPA, MBD and HLA-B7.1 region which induces additional T-cell activation.

example 1

Cloning and expression of the fusion construct SPA-5G-Gephyrin C DNA for gephyrin is cloned by PCR, or the Homo sapiens gephyrin (GPH) m RNA is cloned by RT-PCR. Data from the GPH mRNA sequence are available on the Internet at the National Center for Biotechnology information, NIH, Bethesda MD 208 94, USA, as well as on the internet page of NCBI (https: // www.ncbi.nlm.nih.gov:80/entez/ ... eotide ...).

C DNA for SPA ligated with the primer containing the information for the five-gyzine spacers also becomes cloned with PCR.

The Fusion protein is composed of PCR products. The PCR primers are designed to contain restriction sites on 5 'and 3' ends, for later Perform ligation steps. The 5 'and 3' ends of the gephyrin PCR product contain Bam HI and Hind III restriction sites. The 5 'and 3' ends of the SPA-PCR product contain Xmnl and Bg III restriction sites.

To For amplification and purification, the PCR products are PCR II vectors ligated. Positive clones are screened by plasmids right mass identified. The clones are made by DNA sequencing or checked by standard methods or approved.

The Gephyrin PCR product is released from PCR II by restrictive cleavage out through Bam HI and Hind III, and becomes SPA PCR product cut out of the PCR II by Xmn I and Bg III.

ligation the gephyrin and SPA segments into the pMal-c2 expression vector under standard conditions. The pMal-c2 vector is labeled Bam H I and Hind 3 treated. The gephyrin segment is going through this treatment into the pMal-c2 ligated.

PMAL-c2 gephyrin is cut with X mnl and Bam HI and cut in to SPA segment to ligate.

Ligation buffer is prepared from 66 mM Tris PH 7.6, 5 mM MgCl2, 5 mM DTT and 1 mM ATP, and T4 DNA ligase (20 microliters total). The ligation is at 14 ° C carried out.

The Ligation product is transformed into E coli, expressed and completed cleaned.

Claims (12)

Fusion proteins, characterized in that they contain antibody binding and Mikrotubulibinderegionen. Fusion proteins according to claim 1, characterized by immune-triggering Regions. Fusion proteins according to claims 1 and 2, characterized through long and very long spacer regions. Fusion proteins according to claims 1-3, characterized by membrane penetration domains preferably at the N-terminus of the long or super-long spacer regions. Fusion proteins according to claims 1-4, characterized by at least a nucleic acid binding region. Fusion proteins according to claims 1-5, characterized by at least a polysaccharide binding region. Fusion proteins according to claims 1-5, characterized in that the antigen preferably comprises binding regions following proteins: EGF, FGF, CSF, MGF, IL-15. Fusion proteins according to claims 1-6, characterized by GFP or another fluorescent region. Fusion proteins according to claims 1-7, characterized by at least a GST Histag or another region to carry out the Affinity purification. Fusion proteins according to claims 1-8, characterized by at least a hinge region, preferably a five-glycine region. Fusion proteins according to claims 1-9, characterized that antibody binding regions preferably bind IgG namely Fc regions of IgG and bind preferably from the following regions: staphylococcal protein Aq (SPA), extracellular Region of the Fc receptor CD 64 and others. Regions are selected can. Fusion proteins according to claims 1-10, characterized that the microtubule binder regions preferably consist of the following proteins: Gephyrin, Tau, MAP, MID-1 selected become. Nucleic acid sequences, DNA vectors, cloning and expression systems for the fusion proteins according to the claims 1-11.