Single-chain Multiple Antigen-binding Molecule, Its Preparation and Use

Abstract

The present invention relates to a single-chain, multiple antigen-binding molecule with diverse variable domains of a heavy and of a light chain of an immunoglobulin, which are connected in the form of a VH-VL construct, which are in turn connected together via a peptide, and to the preparation and use thereof as pharmaceutical or diagnostic aid.

Description

CROSS-REFERENCE TO A RELATED APPLICATION[0001]This application is a continuation of co-pending application U.S. Ser. No. 10 / 883,472, filed Jun. 30, 2004; which is a continuation of application U.S. Ser. No. 09 / 288,719, filed Apr. 9, 1999, now U.S. Pat. No. 6,759,518; which claims priority to foreign patent applications DE 198 16 141.7, filed Apr. 9, 1998 and DE 198 27 239.1, filed Jun. 18, 1998; all of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates to a single-chain multiple antigen-binding molecule (“ScMAB”) with diverse variable domains of a heavy and of a light chain of an immunoglobulin, which are connected in the form of a VH-VL construct, which are in turn connected together via a peptide; to nucleic acid molecules encoding ScMAB; to a vector comprising the nucleic acid molecule; to cells comprising the vector; and to the preparation and use thereof in pharmaceuticals or diagnostics.BACKGROUND OF THE INVEN...

AI Technical Summary

Benefits of technology

[0123]A considerable advantage of the present invention is that the linkage of the individual components favours heterodimeric association so that there is predominantly formation of single-chain multiple antigen-binding molecules. There is also a reduction in the dissociation of the dimers, as has been shown for scFv fragments (see, for example, Glockshuber et al., Biochem. 29, 1362-1367, 1990). It is thus possible to prepare the molecules according to the invention with less complexity and in a more homogeneous form than the so-called “diabodies”, even if the latter are disulfide-stabilized or in the form of “knob into hole diabodies”.

[0124]In addition, only one signal sequence and one ribosome binding site (RBS) are necessary for preparing the molecule according to the invention. In contrast to this, a signal sequence and an RBS are required for each chain in the “diabodies” according to the invention. Another advantage of the present invention is that with the molecules according to the invention there is expression of equimolar amounts of the variable domains, whereas on expression of the two chains of “diabodies” there may be production of non-equimolar amounts and thus the proportion of non-functional homodimers is increased.

[0125]Another advantage of the molecule according to the invention is that it can be expressed simply and in functional form both in bacteria and in yeasts, baculoviruses and other eukaryotic cells. Moreover, besides secretion of the molecules according to the invention, there is also the possibility of expressing them inside cells or in association with membranes (see, for example, Biocca & Cattaneo, Trends Cell Biol. 5, 248-252 (1995)).

[0126]Another advantage of the molecules according to the invention is that they can be employed widely both as diagnostic aid, and as drug for the prophylaxis and / or therapy of a disorder, like bispecific antibodies.

[0127]The genes of effectors and promoter sequences are to be selected with a view to the desired use and taking account of the target cell to be transduced. For example, the combinations of promoter sequences and genes for effectors to be chosen for the following disorders are the following:Use of ScMAB in Tumor Therapy

[0128]The target cells for tumor therapy are, for example, proliferating endothelial cells, or the stroma cells and muscle cells adjacent to the endothelial cell, or tumor cells or leukemia cells, the promoters used are, for example, endothelial cell-specific and cell cycle-specific, or cell-nonspecific or muscle cell-specific and cell cycle-specific, or tumor cell-specific (solid tumors, leukemias) and cell cycle-specific promoters, and the effectors used are, for example, the following genes:

Problems solved by technology

There are, however, a number of drawbacks associated with this approach.

In this case, however, there is no selection for the desired heterodimeric molecules, so that mainly bivalent homodimers are produced in this way.

However, this results in both homo- and heterodimers and, when expressed in bacterial cells, non-functional aggregates are produced.

However, in some of these cases non-functional associations of the four variable domains may also occur.

However, preparation thereof is associated with considerable complexity.

In addition, genetic engineering expression of a bispecific “diabody” requires a signal sequence and a ribosome binding site for each chain, which is very complicated.

Although non-equimolar amounts of the variable domains may be expressed, this increases the proportion of non-functional homodimers.

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