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Pretargeting & Crosslinking

Morpholinos in pretargeting and crosslinking applications

"Pretargeting" is the use of a Morpholino conjugated to an antibody to bind a particular antigen (e.g. a tumor marker) followed later by a complementary Morpholino conjugated to a radioisotope or other effector molecule. The high molecular mass antibody conjugate clears the blood slowly, but does not damage the body during its residence in the blood. The low molecular mass Morpholino-cargo conjugate binds rapidly with good affinity to the complementary Morpholino on the antigen-bound antibody, but remaining free Morpholino-cargo conjugate clears the blood quickly. If a radio-labeled oligo is used this keeps the radiation dose well below what it would be if a slowly-clearing radio-labeled antibody were used, but provides the binding specificity of an antibody-based targeting method. Non-radioactive cargos can be delivered in enhanced quantity by attaching numerous Morpholino molecules to the pretargeting antibody

Crosslinking involves treatment with Morpholino-linked antibodies that bind a surface marker of a cell, then following that with complementary Morpholinos linked to a polymer scaffold. This causes aggregation of the surface marker in small regions of the cell surface as the antibody-linked Morpholinos bind the scaffold-linked complementary Morpholinos, in some cases increasing apoptosis.

References

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Pretargeting for delivery of CD20 to induce apoptosis

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Liu Y, Cheng D, Liu X, Liu G, Dou S, Xiao N, Chen L, Rusckowski M, Hnatowich DJ. Comparing the intracellular fate of components within a noncovalent streptavidin nanoparticle with covalent conjugation. Nucl Med Biol. 2012 Jan;39(1):101-7. doi: 10.1016/j.nucmedbio.2011.06.006. Epub 2011 Sep 29.

Liu G, Dou S, Cheng D, Leif J, Rusckowski M, Streeter PR, Schultz LD, Hnatowich D, Greiner D. Human islet cell MORF/cMORF pretargeting in a xenogeneic murine transplant model. Mol Pharm. 2011 Jun 6;8(3):767-73. doi: 10.1021/mp100382m. Epub 2011 Apr 21.

Liu G, Dou S, Chen X, Chen L, Liu X, Rusckowski M, Hnatowich DJ. Adding a clearing agent to pretargeting does not lower the tumor accumulation of the effector as predicted. Cancer Biother Radiopharm. 2010 Dec;25(6):757-62. doi: 10.1089/cbr.2010.0800.

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Liu G, Dou S, Liu Y, Liang M, Chen L, Cheng D, Greiner D, Rusckowski M, Hnatowich DJ. Unexpected side products in the conjugation of an amine-derivatized morpholino oligomer with p-isothiocyanate benzyl DTPA and their removal. Nucl Med Biol. 2011 Feb;38(2):159-63. doi: 10.1016/j.nucmedbio.2010.08.008. Epub 2010 Oct 27.

Liu G, Dou S, Rusckowski M, Greiner D, Hnatowich D. Preparation of (111)In-DTPA morpholino oligomer for low abdominal accumulation. Appl Radiat Isot. 2010 Sep;68(9):1709-14. doi: 10.1016/j.apradiso.2010.03.002.

Cheng D, Wang Y, Liu X, Pretorius PH, Liang M, Rusckowski M, Hnatowich DJ. Comparison of (18)F PET and (99m)Tc SPECT Imaging in Phantoms and in Tumored Mice. Bioconjug Chem. 2010 Aug 18;21(8):1565-70. doi: 10.1021/bc1001467.

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Liu X, Nakamura K, Wang Y, Cheng D, Liang M, Xiao N, Chen L, Rusckowski M, Hnatowich DJ. Auger-mediated cytotoxicity of cancer cells in culture by an 125I-antisense oligomer delivered as a three-component streptavidin nanoparticle. J Biomed Nanotechnol. 2010 Apr;6(2):153-7.

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Liu G, Cheng D, Dou S, Chen X, Liang M, Pretorius PH, Rusckowski M, Hnatowich DJ. Replacing 99mTc with 111In improves MORF/cMORF pretargeting by reducing intestinal accumulation. Mol Imaging Biol. 2009 Sep-Oct;11(5):303-7. doi: 10.1007/s11307-009-0209-0. Epub 2009 Mar 27.

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Liu X, Wang Y, Nakamura K, Kawauchi S, Akalin A, Cheng D, Chen L, Rusckowski M, Hnatowich DJ. Auger Radiation-Induced, Antisense-Mediated Cytotoxicity of Tumor Cells Using a 3-Component Streptavidin-Delivery Nanoparticle with 111In. J Nucl Med. 2009 Apr;50(4):582-90. doi: 10.2967/jnumed.108.056366. Epub 2009 Mar 16.

Chen X, Dou S, Liu G, Liu X, Wang Y, Chen L, Rusckowski M, Hnatowich DJ. Synthesis and in Vitro Characterization of a Dendrimer-MORF Conjugate for Amplification Pretargeting. Bioconjug Chem. 2008 Jul 23. [Epub ahead of print].

Liu G, Dou S, Rusckowski M, Hnatowich DJ. An experimental and theoretical evaluation of the influence of pretargeting antibody on the tumor accumulation of effector. Mol Cancer Ther. 2008 May;7(5):1025-32.

Liu X, Wang Y, Nakamura K, Kubo A, Hnatowich DJ. Cell studies of a three-component antisense MORF/tat/Herceptin nanoparticle designed for improved tumor delivery. Cancer Gene Ther. 2008 Feb;15(2):126-32. Epub 2007 Dec 14.

Wang Y, Nakamura K, Liu X, Kitamura N, Kubo A, Hnatowich DJ. Simplified Preparation via Streptavidin of Antisense Oligomers/Carriers Nanoparticles Showing Improved Cellular Delivery in Culture. Bioconjug Chem. 2007 Jul-Aug;18(4):1338-43. Epub 2007 Jul 3.

Zhang S, Liu G, Liu X, Yin D, Dou S, He J, Rusckowski M, Hnatowich DJ. Comparison of Several Linear Fluorophore- and Quencher-Conjugated Oligomer Duplexes for Stability, Fluorescence Quenching, and Kinetics in Vitro and in Vivo in Mice. Bioconjug Chem. 2007 May 19; [Epub ahead of print].

He J, Liu G, Dou S, Gupta S, Rusckowski M, Hnatowich D. An Improved Method for Covalently Conjugating Morpholino Oligomers to Antitumor Antibodies. Bioconjug Chem. 2007 May-Jun;18(3):983-8. Epub 2007 Mar 27.

Liu G, Dou S, Yin D, Squires S, Liu X, Wang Y, Rusckowski M, Hnatowich DJ. A novel pretargeting method for measuring antibody internalization in tumor cells. Cancer Biother Radiopharm. 2007 Feb;22(1):33-9.

Liu G, Dou S, He J, Liu X, Rusckowski M, Hnatowich DJ. Predicting the biodistribution of radiolabeled cMORF effector in MORF-pretargeted mice. Eur J Nucl Med Mol Imaging. 2007 Feb;34(2):237-46.

He J, Rusckowski M, Wang Y, Dou S, Liu X, Zhang S, Liu G, Hnatowich DJ. Optical Pretargeting of Tumor with Fluorescent MORF Oligomers. Mol Imaging Biol. 2007 Jan-Feb;9(1):17-23.

Liu G, Dou S, Mardirossian G, He J, Zhang S, Liu X, Rusckowski M, Hnatowich DJ. Successful Radiotherapy of Tumor in Pretargeted Mice by 188Re-Radiolabeled Phosphorodiamidate Morpholino Oligomer, a Synthetic DNA Analogue. Clin Cancer Res. 2006 Aug 15;12(16):4958-4964.

Liu G, Dou S, He J, Yin D, Gupta S, Zhang S, Wang Y, Rusckowski M, Hnatowich DJ. Radiolabeling of MAG(3)-morpholino oligomers with (188)Re at high labeling efficiency and specific radioactivity for tumor pretargeting. Appl Radiat Isot. 2006 Sep;64(9):971-8. Epub 2006 May 30.

He J, Liu X, Zhang S, Liu G, Hnatowich DJ. Affinity enhancement bivalent morpholinos for pretargeting: surface plasmon resonance studies of molecular dimensions. Bioconjug Chem. 2005 Sep-Oct;16(5):1098-104.

Liu G, He J, Dou S, Gupta S, Rusckowski M, Hnatowich DJ. Further investigations of morpholino pretargeting in mice-establishing quantitative relations in tumor. Eur J Nucl Med Mol Imaging. 2005 Sep;32(9):1115-23.

He J, Liu G, Vanderheyden JL, Dou S, Mary R, Hnatowich DJ. Affinity enhancement bivalent morpholino for pretargeting: initial evidence by surface plasmon resonance. Bioconjug Chem. 2005 Mar-Apr;16(2):338-45.

He J, Liu C, Vanderheyden JL, Liu G, Dou S, Rusckowski M, Hnatowich DJ. Radiolabelling morpholinos with 188Re tricarbonyl provides improved in vitro and in vivo stability to re-oxidation. Nucl Med Commun. 2004 Jul;25(7):731-736.

He J, Liu G, Gupta S, Zhang Y, Rusckowski M, Hnatowich DJ. Amplification targeting: a modified pretargeting approach with potential for signal amplification-proof of a concept. J Nucl Med. 2004 Jun;45(6):1087-95.

Liu G, He J, Dou S, Gupta S, Vanderheyden JL, Rusckowski M, Hnatowich DJ. Pretargeting in tumored mice with radiolabeled morpholino oligomer showing low kidney uptake. Eur J Nucl Med Mol Imaging. 2004 Mar;31(3):417-24. Epub 2003 Dec 23.

He J, Liu G, Zhang S, Rusckowski M, Hnatowich DJ. Pharmacokinetics in mice of four oligomer-conjugated polymers for amplification targeting. Cancer Biother Radiopharm. 2003 Dec;18(6):941-7.

He J, Liu G, Zhang S, Vanderheyden JL, Liu N, Liu C, Zhang Y, Gupta S, Rusckowski M, Hnatowich DJ. A comparison of in vitro and in vivo stability in mice of two morpholino duplexes differing in chain length.. Bioconjug Chem. 2003 Sep-Oct;14(5):1018-23.

Liu G, Liu C, Zhang S, He J, Liu N, Gupta S, Rusckowski M, Hnatowich DJ.. Investigations of 99mTc morpholino pretargeting in mice.. Nucl Med Commun 2003 Jun;24(6):697-705.

Liu C, Liu G, Liu N, Zhang YM, He J, Rusckowski M, Hnatowich DJ. Radiolabeling morpholinos with (90)Y, (111)In, (188)Re and (99m)Tc. Nucl Med Biol 2003 Feb;30(2):207-14.

Liu G, He J, Zhang S, Liu C, Rusckowski M, Hnatowich DJ. Cytosine residues influence kidney accumulations of 99mTc-labeled morpholino oligomers. Antisense Nucleic Acid Drug Dev. 2002 Dec;12(6):393-8.

Liu G, Zhang S, He J, Liu N, Gupta S, Rusckowski M, Hnatowich DJ.. The influence of chain length and base sequence on the pharmacokinetic behavior of 99mTc-morpholinos in mice. Q J Nucl Med. 2002 Sep;46(3):233-43.

Liu G, Zhang S, He J, Zhu Z, Rusckowski M, Hnatowich DJ. Improving the Labeling of S-Acetyl NHS-MAG(3)-Conjugated Morpholino Oligomers. Bioconjug Chem. 2002 Jul 17;13(4):893-897.

Liu G, Mang'era K, Liu N, Gupta S, Rusckowski M, Hnatowich DJ. Tumor Pretargeting in Mice Using (99m)Tc-Labeled Morpholino, a DNA Analog. J Nucl Med. 2002 Mar;43(3):384-391.

Mang'era KO, Liu G, Yi W, Zhang Y, Liu N, Gupta S, Rusckowski M, Hnatowich DJ. Initial investigations of 99m Tc-labeled morpholinos for radiopharmaceutical applications. Eur J Nucl Med 2001 Nov;28(11):1682-9.