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"Cell Separation Method Using a Release System for Cell-Antibody-Substrate Conjugates Containing a Polyethylene Glycol Spacer Unit" in Patent...

May 13, 2014



"Cell Separation Method Using a Release System for Cell-Antibody-Substrate Conjugates Containing a Polyethylene Glycol Spacer Unit" in Patent Application Approval Process

By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Brieden, Jennifer (Ennepetal, DE); Dose, Christian (Bergisch Gladbach, DE), filed on October 2, 2013, was made available online on May 1, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Miltenyi Biotec GmbH).

This patent application is assigned to Miltenyi Biotec GmbH.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The present invention relates to a release system for cell-antibody-substrate conjugates; a method to selectively dissociate the conjugates, and a cell separation method using the release system.

"The ability of biotin to bind streptavidin, avidin, and other biotin-binding molecules has been exploited for several decades, because of the high affinity, specificity, and broad applicability of this system.

"To improve the release properties of biotin/streptavidin affinity systems, chemically modified biotin and streptavidin derivatives have been introduced, wherein only one or even both binding partners were modified. Such modifications lower the stability of the biotin/streptavidin complex by several orders of magnitude and thereby facilitate the dissociation of the two binding partners (see for example US 20080255004). Furthermore, mutated streptavidin proteins have been developed with reduced affinity for biotin or its analogues (M. Qureshi et al, J. Biol. Chem. 276 (2001) 46422-46428; T. Sano et al, Proc. Natl. Acad. Sci. USA 92 (1995) 3180-3184). U.S. Pat. No. 5,506,121 discloses so called 'strept-tags' i.e. peptides with reduced binding affinity to streptavidin.

"The modification of biotin or streptavidin is laborious. Since many reagents for the coupling of biotin with substrates, like antibodies, are commercially available, it is widely practiced to use unmodified biotin as labeling agent.

"In this respect, U.S. Pat. No. 5,215,927 describes the isolation of target cells by contacting the desired cell population with a monoclonal antibody and subsequent incubation with a biotinylated anti-species immunoglobulin directed to the specific monoclonal antibody. The mixture is separated over a solid phase comprising immobilized avidin as biotin-binding molecule, which facilitates the immobilization of the target cells and separation of unlabeled targets. The desired cells are subsequently released by mechanical agitation to disrupt the immobilized complex.

"The use of release mechanisms mediated by unselective enzyme degradation, chemical reactions, intense mechanical forces, high temperature, or strong saline conditions are undesirable for the separation of living cells, because it is important to preserve the cells' integrity and viability. Accordingly, mechanisms are desired that allow a rapid and selective release of the target cells at physiological conditions.

"As an alternative to enzymatic or chemical cleavage or the use of modified biotin/streptavidin molecules, biotin/streptavidin systems can also be cleaved by a ligand competition mechanism. For example, a biotinylated molecule can be released from a streptavidin-support by adding an excess of free biotin, thereby replacing the biotinylated molecule.

"Methods based on the competition of free biotin or streptavidin against the respective counterpart (streptavidin or biotin) are known in numerous variants. James Hirsch et al. give an overview of these techniques in Analytical Biochemistry 308 (2002) 343-357.

"Furthermore, Ke Shan et al. disclose in 'Avidin-Biotin-PEG-CPA Complexes as Potential EPR-Directed Therapeutic Protein Carriers: Preparation and Characterization', Bioconjugate Chemistry, vol. 18, no. 5, 1 Sep. 2007 (2007-09-01), pages 1644-1650 and 'Intermolecular interaction of avidin and PEGylated biotin', Bioconjugate Chemistry, vol. 18, no. 6, 21 Nov. 2007 (2007-11-21), pages 2109-2114, the use of biotin for dissociation of the Avidin-Biotin-PEG-enzyme-complex.

"The competition reaction of free biotin/streptavidin against the respective bound counterpart is disclosed in WO 92/16841 for analytical means. WO 92/16841 describes inter alia a method for detecting a reporter molecule, which is specifically bound to an analyte and an insoluble phase via a streptavidin/biotin-binding system. After the work-up procedure, the streptavidin/biotin binding system is cleaved by a displacement ligand and the released analyte is analytically detectable via the reporter molecule.

"US 2008/0255004, U.S. Pat. No. 6,869,606, and U.S. Pat. No. 4,656,252 disclose biotinylated antibodies comprising modified biotin, wherein the biotin moiety and the antibody moiety of the biotinylated antibodies are separated by a spacer group consisting of an aryl, alkyl, or aminocaprolic acid group. The use of hydrophobic aryl or alkyl groups is expected to cause agglomeration in aqueous solutions. Since physiological conditions of biological systems usually require aqueous solutions, agglomeration is an eminent problem for techniques utilizing rather hydrophobic substances. Spacer molecules derived from amino caprolic acid (so called 'LC linker') possess a linear alkyl chain with residues that support homo- or heterofunctional bioconjugation chemistries.

"U.S. Pat. No. 5,518,882 discloses a similar method, wherein target cells are bound to a solid support, for example magnetic particles. This allows a further enrichment by applying a magnetic field, which immobilizes the target cells coupled to the magnetic beads. The target cells can be released from the particles by cleaving the biotin-binding system with a displacement ligand. Preferably, the conjugate '(magnetic bead)-antibiotin-biotin-antibody-cell' is cleaved by adding free streptavidin in access, resulting in a '(magnetic bead)-antibiotin' and a 'streptavidin-biotin-antibody-cell' conjugate.

"In general, a competition reaction, i.e. the displacement of a first ligand with a second ligand, will only proceed until the equilibrium between the kinetics of the binding reaction of the first and second ligand is reached. The equilibrium depends on the respective binding forces and concentrations of the ligand and the thermodynamic conditions of the reaction. The known competition reactions to displace biotin by streptavidin therefore lead either to an uncompleted release or are difficult to control since the underlying kinetics are usually not known.

"The present release systems are not fast or reliable enough for a process involving labeling of living cells, cell separation or detection and unlabeling of the target cells."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "It was therefore an object of the present invention to provide a reliable and highly efficient release system comprising a biotinylated antibody for detection of living cells and its use in a cell separation method.

"It is found that the binding affinity of a labeled binding partner targeted to the biotin moiety of a biotinylated antibody can be adjusted by placing a biocompatible spacer molecule between the biotin moiety and the antibody, wherein the labeled binding partner is provided with a detection means. With such a conjugate, cells can be first labeled for identification or isolation and after identification or isolation, the label can be removed to unlabel the desired target cells.

"Disclosed here is a releasable conjugate comprising a biotinylated ligand having a biotin moiety, a ligand moiety (Ligand.sub.1) and a biotin-binding molecule (bbm) bound to the biotin moiety of the biotinylated ligand wherein the biotin moiety and the ligand moiety of the biotinylated ligand are separated by a spacer group consisting of polyethylene glycol. Release agents and methods for the conjugate are also disclosed.

"These and other features and advantages are described in, or are apparent from, the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

"Various exemplary details are described with reference to the following figures, wherein:

"FIG. 1 shows conjugates containing Biotin (1), LC-Biotin (2), LC-LC-Biotin (3), and PEO-Biotin (4);

"FIG. 2 shows the mean fluorescence intensities that have been achieved with different anti-CD8-Biotin conjugates as a function of the B/P ratio;

"FIG. 3 shows the relative fluorescence signal of CD8 positive cells in the presence of different anti-CD8-Biotin conjugates;

"FIG. 4 shows the relative fluorescence signal of CD8 positive cells with different auxiliary release agents;

"FIG. 5 illustrates the relative dissociation of the anti-CD8-PEO-Biotin/anti-Biotin-PE system compared to the anti-CD8-LC-LC-Biotin/anti-Biotin system in the presence of streptavidin;

"FIG. 6 illustrates the dissociation of the anti-CD8-PEO-Biotin/anti-Biotin-PE system compared to the anti-CD8-LC-LC-Biotin/anti-Biotin system in the presence of APC-streptavidin;

"FIG. 7 is a comparison of magnetically labeled anti-CD8-PEO-Biotin/anti-Biotin system to the anti-CD8-LC-LC-Biotin/anti-Biotin system with the addition of biotin as release agent;

"FIG. 8 is a comparison of the magnetically labeled anti-CD8-PEO-Biotin/anti-Biotin system to the anti-CD8-LC-LC-Biotin/anti-Biotin system in the presence of streptavidin; and

"FIG. 9 is a comparison of the magnetically labeled anti-CD8-PEO-Biotin/anti-Biotin system to the anti-CD8-LC-LC-Biotin/anti-Biotin system in the presence of APC streptavidin."

URL and more information on this patent application, see: Brieden, Jennifer; Dose, Christian. Cell Separation Method Using a Release System for Cell-Antibody-Substrate Conjugates Containing a Polyethylene Glycol Spacer Unit. Filed October 2, 2013 and posted May 1, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2429&p=49&f=G&l=50&d=PG01&S1=20140424.PD.&OS=PD/20140424&RS=PD/20140424

Keywords for this news article include: Antibodies, Biotin, Alkenes, Therapy, Polyenes, Chemistry, Immunology, Hydrocarbons, Streptavidin, Micronutrient, Blood Proteins, Immunoglobulins, Organic Chemicals, Bacterial Proteins, Diet and Nutrition, Miltenyi Biotec GmbH, Polyethylene Glycols.

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Source: Life Science Weekly


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