Chemical Biology

Wagner Lab

Chemically Self-Assembled Nanostructures (CSANs)

Our laboratory has developed a method for the engineering and preparation of chemically self-assembling nanorings (CSANs) that can display targeting peptides and proteins, such as single chain antibodies (scFvs). CSANs are prepared by taking advantage of the power of high affinity chemically-induced dimerization.

Previously, we had found that when mixed with a dimer of the dihydrofolate reductase (DHFR) inhibitor methotrexate, bisMTX, DHFR forms highly robust DHFR dimers with an affinity of approximately 10-11M. If one DHFR was recombinantly fused to another DHFR by an encoded linker peptide (yielding DHFR-DHFR or DHFR2), we observed their spontaneous and rapid self-assembly into CSANs, whose diameter was dependent on the length and composition of the linker peptide (13- amino acid linker = dimer, 1- amino acid linker = octamer).

The rings exhibited high stability with Tms ranging from 63-66oC and could be easily disassembled with the non-toxic DHFR inhibitor trimethoprim. Single molecule experiments have also confirmed that even at picomolar concentrations, nearly 70% of the nanorings remain intact. Since the CSANs exhibited the properties of a stable scaffold, we have functionalized them by fusing sc-Fvs to the C-terminus of DHFR2. The resulting monovalent, bivalent or octavalent targeted CSANs were found to tightly bind to cell surfaces and depending on the targeted receptor undergo rapid internalization.

We have used this feature to prepare targeted CSANs that can be used for drug and nucleic acid delivery, as well as for fluorescence and PET imaging. We have further expanded the capabilities of the CSANSs by preparing bispecific CSANs that enable the targeting of effector T-cells to cancer cells, leading to in vivo tumor erradication. In addition, we have developed a new generation of CSANs for the rapid modification of cell surfaces, thus enabling us to design and engineer cell-cell interactions. We are currently applying this later approach as an alternative to current genetic based methods for the development of adoptive immune cell therapies and stem cell base regenerative medicine approaches.

Key Accomplishments:

Developed nanomaterial produced using principles of chemically induced protein assembly.
Developed protein nanomaterial capable of targeted nucleic acid delivery.
Developed protein nanomaterial capable of targeted PET imaging.
Developed nanomaterial designed for engineering cell-cell interactions.
Developed generalizable and reversal system for the rapid non-genetic modification of T-cells in vivo.
Developed generalizable and reversal approach for the targeting to T-cells of primary and tumor stem like cells, leading to tumor eradication.
Developed generalizable and reversal approaches using hydrophobic insertion of a nanomaterial for the modification and engineering of any cell surface.
Developed quantitative analysis of the relationship between ligand binding, valency and cell surface antigen expression on multivalent particle homing in vitro and in vivo.

Citations

Fegan, Adrian, Kumarapperuma, Sidath, and Wagner, Carston R. “Chemically Self-assembled Antibody Nanostructures as Potential Drug Carriers”, Mol. Pharmaceutics 9, 3218–3227 (2012)

Gabrielse, Kari, Gangar, Amit, Kumar, Nigam, Lee, Jae Chul, Fegan, Adrian, Shen, Jing Jing, Li, Qing, Vallera, Daniel A. and Wagner, Carston R., “Reversible Re-programming of Cell-Cell Interactions”, Angewante Chemie (International. Ed.) 53, 5112-5116 (2014).

Shen, Jingjing, Vallera, Daniel A., and Wagner, Carston R., “Prosthetic Antigen Receptors”, J. Amer. Chem. Soc., 137, 10108-10111 (2015).

Shah, Rachit, Petersburg, Jacob, Gangar, Amit C., Fegan, Adrian, Wagner, Carston R., and Kumarapperuma, Sidath C., “In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures”, Molecular Pharmaceutics, 13, 2193–2203 (2016).

Petersburg, Jacob R., Jingjing Shen, Jingjing, Csizmar, Clifford M., Murphy, Katherine A Spanier, Justin, Gabrielse, Kari, Griffith, Thomas S., Fife, Brian and Wagner, Carston R., “Eradication of Established Tumors by Chemically Self-Assembled Nanoring (CSAN) Labelled T-Cells”, ACS Nano, ASAP (2018).

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