Macromolecular conjugates for visualization and separation of proteins and cells

Abstract

Macromolecular water-soluble conjugates based on synthetic copolymers to which at least one affinity tag, at least one imaging probe, and at least one targeting ligand are bound via covalent bonds. The macromolecular conjugate may be used in identification, visualization, quantification or isolation of proteins and/or cells. The targeting ligand may be attached to the synthetic copolymer via a flexible linker.

Description

FIELD OF ART[0001]The invention describes synthetically prepared macromolecules having properties of monoclonal antibodies, said macromolecules being capable of replacing the use of antibodies in scientific research, in diagnostics, in biochemical investigations and for the preparation of targeted drugs. These synthetic macromolecules, targeted and binding specifically to certain proteins, are suitable for the visualization, identification and isolation of biomolecules and / or cells in biochemistry, molecular biology and medicine and as targeting ligands in the pharmacy and diagnostics.BACKGROUND ART[0002]The discovery and the subsequent use of monoclonal antibodies allowing detection and specific binding of biologically important molecules caused a revolution in biochemistry and molecular biology as well as in the diagnosis and treatment of numerous serious diseases. In science, this discovery led to the development of many important techniques today considered routine, such as West...

AI Technical Summary

Benefits of technology

[0018]Targeting ligand may be attached to the synthetic copolymer via a flexible linker, based on e.g. (oligo)polyethylene glycol, peptide, nucleic acid or oligosaccharide. The linker allows inhibitor binding to the active site of the enzyme so as to avoid steric hindrance of the binding by the polymer and time such linker allows targeting enzymes with active site hidden in the binding cavity of the enzyme. Preferably, the linker is selected from the group consisting of linkers based on polyethylene glycol, peptide, preferably a peptide having a molecular weight from 100 to 5000 g / mol, or nucleic acid, preferably a nucleic acids comprising 1 to 40 nucleotides, or oligosaccharide, preferably an oligosaccharide containing 1 to 40 monosaccharides.

[0019]Compared to currently used antibodies, synthetic molecules provided by this invention provide several advantages. Preparation of polymeric conjugates is inexpensive, and in comparison to antibodies, if there is an inhibitor of the enzyme, conjugates are also relatively easily prepared. Polymeric conjugates are chemically substantially more stable and their solutions can be repeatedly frozen and thawed without significant influence on their ability to bind the enzyme. One of the biggest advantages of these conjugates is that due to the present inhibitor they bind to the active site of the enzyme and thus bind only to enzymatically active form of the enzyme, i.e. always to a native protein. Antibodies lack this ability. Another advantage is the “non-biological” origin of the polymeric backbone—in many methods with complex matrices (e.g. immunoprecipitation from blood plasma, etc.) there is competition between endogenous antibodies or other proteins and the respective antibodies used in the experiment, which often leads to a reduction in the success of the experiment down to impracticability (e.g. frequent emergence of false positive results in ELISA).

Problems solved by technology

First, the production of antibodies is very expensive.

Like other proteins, antibody molecules are susceptible to degradation: generally they must be stored at low temperatures, and if necessary, frozen in aliquots.

Their repeated thawing often leads to loss of their ability to bind a given antigen.

Another disadvantage is their own creation and method of preparation, since the immunization of an animal may not always lead to successful production of antibodies—they often may not be produced at all, or can be non-specific to the antigen.

Another disadvantage is the fact that for a close group of enzymes (ie. homologs, either paralogs or orthologs), it is often impossible to use the same antibody (recognizing native proteins) due to differences in amino acid residues on their surface.

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