Overview: The research interests of the Liu laboratory at UNC Eshelman School of Pharmacy and Carolina Center for Genome Sciences focus on the development and application of novel drug target-binding affinity molecules by integrating directed molecular selection and evolution, ligand design and engineering, in vitro cellular and signaling characterization, and in vivo therapeutic efficacy studies in tumor mouse models.
Types of biological molecules the Liu lab is developing and studying:
- Conformation- or ligand-specific drug target binders based on polypeptide domains or motifs
- Multi-functional recombinant proteins or cell-surface chimeric antigen receptors
- Nuclease-resistant 2’-fully modified RNA aptamers
The technology platforms: Directed evolution of polypeptides and polynucleotides in test tubes from combinatorial libraries with high diversity is a powerful platform that allows for quick development of novel biological molecules that have desired chemical, biological, and pharmaceutical properties. The Liu lab has been developing and/or using novel display and selection platforms, including the following:
- mRNA Display: mRNA display, developed by the Szostak lab at MGH/Harvard, is a totally in vitro polypeptide selection platform in which each polypeptide is covalently linked with its coding mRNA, allowing for efficient selection of natural and unnatural polypeptide libraries with up to 100-trillion unique sequences. The Liu lab pioneered the development of mRNA display and its application in scanning natural proteomes and synthetic domain libraries for proteins with desired biological activities.
- Yeast Surface Display: Yeast surface display, developed by the Wittrup lab at MIT, is a cellular-based polypeptide selection platform in which a protein of interest is displayed on the surface of yeast cells, allowing quantitative screening of protein libraries with up to billion unique sequences through fluorescent-activated cell sorting. The Liu lab combines mRNA display and yeast surface display, aiming at efficient quantitative selection of protein libraries with unusually high diversities.
- SELEX for fmRNA: Compared to polypeptide-based biologics, polynucleotide-based aptamers possess unique properties, including high in vitro evolvability, target-binding affinity, specificity, low immunogenicity and toxicity. We developed the 2’-fmRNA SELEX system that allows systematic evolution of 2′-fully modified RNA aptamers that have superior nuclease and serum stability compared to 2′-partially modified RNA variants. The resulting fmRNA SELEX can be readily performed at molecular (conventional SELEX), cellular (cell SELEX), and whole animal (in vivo SELEX) levels.
- Mouse models for in vivo studies: After extensive characterization and optimization of the selected biologics at the molecular and cellular levels, we investigate their anti-tumor efficacies in mouse models that mimic the human diseases. The tumor models that are currently used in the Liu lab include: for triple-negative breast cancer: orthotopic 4T1 and EMT6 (syngeneic) mouse models; for primary CRC and liver metastasis: subcutaneous, orthotopic, and hemi-spleen CT26 (syngeneic) mouse models; for HCC: orthotopic Hepa1-6 (syngeneic) mouse models, for pancreatic cancer: subcutaneous and orthotopic KPC (syngeneic) mouse models; for primary uveal melanoma and liver metastasis: orthotopic (eye) and hemi-spleen OMM1.3, 92.1, and MP38 (human) mouse models.
Related Publications:
Szostak, J., Roberts, R., and Liu, R. WO/1998/031700 (1998); “Selection of proteins using RNA-protein fusions”.
Shen, X., Valencia, C. A., Szostak, J. W., Dong, B., and Liu, R. (2005) “Scanning the Human Proteome for Calmodulin-binding Proteins”; Proceedings of the National Academy of Sciences of US, 102, 5969-5974. PMID: 15840729.
Ju, W., Valencia, C. A., Pang, H., Ke, Y., Gao, W., Dong, B., and Liu, R. (2007) “Proteome-wide Identification of Member-specific Natural Substrate Repertoire of Caspases”; Proceedings of the National Academy of Sciences of US, 104, 14294-14299. PMID: 17728405.
Cotten, S.W., Zou, J., Valencia, C.A., and Liu, R. (2011) “Selection of Proteins with Desired Properties from Natural Proteome Libraries Using mRNA Display”, Nature Protocols, 6, 1163-1182. PMID: 21799486.
Wang, H. and Liu, R. (2011) “Advantages of mRNA Display Selections Over Other Selection Techniques for Investigation of Protein-Protein Interactions”, Expert Reviews of Proteomics, 8 (3), 335-346. PMID: 21679115.
Wang, H., Vilela, M., Winkler, A., Tarnawski, M., Hartmann, E., Schlichting, I., Yumerefendi, H., Kuhlman, B., Liu, R.*, Danuser, G.*, Hahn, K.* (2016) “LOVTRAP, A Versatile Optogenetic System, Reveals Resonator Motifs in Mammalian Mechano-chemical Signaling Pathways”, Nature Methods, Sep. 2016; 13(9): 755-758. PMID: 27427858.
Friedman, A.D., Kim, D.W., and Liu, R. (2014) “Highly stable aptamers selected from a 2’-fully modified fGmH RNA library for targeting biomaterials”, Biomaterials, 2014, 36(1): 110-123. PMID:25443790.
Current projects in the Liu lab:
Novel biologics with tumor targeting and/or immunomodulatory properties: The modulation of the sophisticated immunosuppression of the tumor microenvironment (TME) has great potential to revolutionize our understanding and the clinical treatment of many types of human cancers as well as other immune-related diseases. Successful targeting of cancer cells and blockade of immunosuppression-related molecules rely on drug target-binding molecules with high affinity and specificity. Using directed molecular evolution and protein display-based engineering, the Liu lab develop novel protein domain-based targeting ligands and immune modulators (inhibitory traps/decoys or stimulatory activators) that potently and specifically bind to the targets of interest at critical sites (active, ligand-binding or ligand/receptor interacting sites). We are particularly interested in targeting tumor surface antigens (i.e. ErbBs or neoantigens), immunosuppression regulators (i.e. ILs, CXCLs, CCLs, and Wnts), immune checkpoints (i.e. PD-1 and its ligands), and signaling molecules in innate immune responses (i.e. STING) that play pivotal roles in the tumorigenesis, metastasis and immunosuppression of different solid tumors. The resulting biologics can serve as tumor-homing or/and immunotherapeutic agents in different formats, including the recombinant proteins, mRNAs delivered by lipid nanoparticles, cDNA delivered by AAV, and cell surface proteins delivered by immune cells (CAR-T).
Related Publications:
Kim, D.W., Friedman, A.D., and Liu, R. (2014) “Tetraspecific ligand for tumor-targeted delivery of nanomaterials”, Biomaterials, 2014, 35(23): 6026-6036. PMID: 24786763.
Goodwin, T., Zhou, Y., Musetti, S., Liu, R.*, Huang, L.* (2016) “Local and Transient Gene Expression Primes the Liver to Resist Colorectal cancer metastasis”, Science Translational Medicine, Nov. 9 2016; 8(364): 364ra153. PMID: 27831902.
Goodwin, T.J., Shen, L., Hu, M., Li, J., Feng, R., Dorosheva, O., Liu, R.*, Huang, L.* (2017) “Liver specific gene immunotherapies resolve immune suppressive ectopic lymphoid structures of liver metastases and prolong survival”, Biomaterials, 2017 Oct;141:260-271. PMID: 28700955.
Shen, L., Li, J., Liu, Q., Song, W., Zhang, X., Tiruthani, K., Hu, H., Das, M., Goodwin, T.J., Liu, R.*, Huang, L.* (2018) “Local Blockade of Interleukin 10 and C-X-C Motif Chemokine Ligand 12 with Nano-Delivery Promotes Antitumor Response in Murine Cancers”, ACS Nano. 2018 Sep 28. doi: 10.1021/acsnano.8b00967. [Epub ahead of print] PMID: 30253648.
Wang, Y., Song, W., Hu, M., An, S., Xu, L., Li, J., Kinghorn, K. A., Liu, R.*, Huang, L.* (2018) “Nanoparticle-mediated HMGA1 Silencing Promotes Lymphocyte Infiltration and Boosts Checkpoint Blockade Therapy for Cancer”, Advanced Functional Materials, 2018, July 24; 28 (36). https://doi.org/10.1002/adfm.201802847.
An, S., Tiruthani, K., Wang, Y., Xu, L., Hu, M., Li, J., Song, W., Jiang, H., Sun, J., Liu, R.*, Huang, L.* (2019) “Locally Trapping the C-C Chemokine Receptor Type 7 by Gene Delivery Nanoparticle Inhibits Lymphatic Metastasis Prior to Tumor Resection”, Small, 2019 Mar;15(9):e1805182. doi: 10.1002/smll.201805182. Epub 2019 Jan 28. PMID: 30690891.
Wang, Y., Tiruthani, K., Hu, M., Zhang, L., Liao, C., Tan, J., Liu, R. “mRNA Delivery of a Bispecific Single Domain Antibody Polarizes Tumor-associated Macrophages and Synergizes the Immune Checkpoint Blockade Immunotherapy”, Under review.
Multi-functional biologics via self-assembly. One effective approach to expanding the application spectrum of biologics and improving their therapeutic efficacies is by tunably integrating multiple functions, including valency, specificity, and potency, into one biological molecule. The Liu lab has been applying the strategies from Mother Nature to develop technology platforms that allow for highly efficient self-assembly of biological molecules with tunable multivalency (i.e. trimeric, tetrameric, pentameric, heptameric) and multispecificity.
Based on the robust trimerization platform we developed, the Liu lab invented a panel of trimeric immune blockers/traps or stimulators against important immunosuppression targets, including antagonistic blockers against immune co-inhibitory PD-1 and PD-L1/2, agonistic stimulators against immune co-stimulatory CD40, OX40, and 4-1BB (and their ligands), TLR4 antagonist (trimeric LPS trap), and Wnt antagonists (Wnt trap and R-spondin trap). Distinctive from PD-1 or PD-L1 mAbs, the pan PD-1 ligand inhibitor (panPD1Linh) developed at Liu lab can simultaneously block all three major immune co-inhibitory interactions, including PD-1/PD-L1, PD-1/PD-L2, and PD-L1/CD80. We perform translational studies of the resulting multi-functional biologics in different syngeneic, orthotopic mouse models, aiming at potent disruption of the immunosuppressive signaling pathways in TME and consequently significant immunotherapeutic efficacy.
Related Publications:
Duan, J., Wu, J., Valencia, C. A., and Liu, R. (2007) “Fibronectin Type III Domain Based Monobody with High Avidity”, Biochemistry, 46 (44), 12656-12664. PMID: 17929945.
Simnick, A.J., Valencia, C.A., Liu, R., Chilkoti, A. (2010) “Morphing Low-Affinity Ligands into High-Avidity Nanoparticles by Thermally Triggered Self-Assembly of a Genetically Encoded Polymer”, ACS Nano. 4(4):2217-27. PMID: 20334355.
Kim, D.K., Yan, Y., and Liu, R. (2012) “Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity”, PLOS ONE, 2012;7(8):e43077. Epub 2012 Aug 9.
Kim, D.W., Kim, S.K, Valencia, C.A., and Liu, R. (2013) “Tribody: Robust Self-assembled Trimeric Targeting Ligands with High Stability and Significantly Improved Target-binding Strength”, Biochemistry, 52(41), 7283-7294. PMID: 24050811.
Kim, D.W., Friedman, A.D., and Liu, R. (2014) “Tetraspecific ligand for tumor-targeted delivery of nanomaterials”, Biomaterials, 2014, 35(23): 6026-6036. PMID: 24786763.
Miao, L., Li, J., Liu, Q., Feng, R., Das, M.C., Lin, M., Goodwin, T., Dorosheva, O., Liu, R.*, Huang, L.* (2017) “Transient and Local Expression of Chemokine and Immune Checkpoint Traps to Treat Pancreatic Cancer”, ACS Nano, 2017 Sep 26;11(9):8690-8706. PMID: 28809532.
Liu, Q., Zhu, H., Tiruthani, K., Shen, L., Chen, F., Gao, K., Zhang, X., Hou, L., Wang, D., Liu, R.*, Huang, L.* (2018) “Nanoparticle-Mediated Trapping of Wnt Family Member 5A in Tumor Microenvironments Enhances Immunotherapy for B-Raf Proto-Oncogene Mutant Melanoma”, ACS Nano, 2018 Feb 27;12(2):1250-1261. doi: 10.1021/acsnano.7b07384. PMID: 29370526.
Song, W., Shen, L., Goodwin, T., Liu, Q., Li, J., Dorosheva, O., Liu, T., Wang, Y., Das, M., Liu, R.*, Huang, L.* (2018) “Synergistic and Low Adverse Effect Cancer Immunotherapy by Immunogenic Chemotherapy and Locally Expressed PD-L1 Trap”, Nature Communications, 2018 Jun 8;9(1):2237. doi: 10.1038/s41467-018-04605-x. PMID: 29884866.
Song, W., Tiruthani, K., Wang, Y., Shen, L., Hu, M., Dorosheva, O., Qiu, K., Kinghorn, K., Liu, R.*, Huang, L* (2018) “Trapping Lipopolysaccharide to Promote Immunotherapy against Colorectal Cancer and Attenuate Liver Metastasis”, Advanced Materials, 2018 Nov 2:e1805007. doi: 10.1002/adma.201805007. [Epub ahead of print] PMID:30387230.
Next generation CAR-T cells. CAR T-cell therapy is a personalized, cell-based treatment that uses in vitro engineered tumor-targeting T cells to recognize and kill cancer cells displaying specific surface antigen. CAR T-cell technology is still at its early stage and major unmet needs should be addressed to fully achieve its long-term potential. The Liu lab addresses the challenges by evolving highly modular non-immunoglobulin chimeric antigen receptors (CARs) and use them to generate next generation CAR-T platforms, including those with tunable biepitopicity, bispecificity, as well as those only activated in the tumor microenvironment.
Related Publications:
Ahn, S., Li, J., Sun, C., Gao, K., Li, H., Savoldo, B., Liu, R.*, Dotti, G.* (2019) “T cells Redirected with Biepitopic and Bispecific Antibody Mimic Receptors for Cancer Immunotherapy”, Cancer Immunology Research, May;7(5):773-783. doi: 10.1158/2326-6066.CIR-18-0636. Epub 2019 Mar 6. PMID: 30842091.
Inhibitory decoys specific to the active forms of GTPases. Galpha-q, or its closely related homolog, Galpha-11, is constitutively active in approximately 83% of all UM cases due to a single mutation that abolishes GTP hydrolysis. Using mRNA display-based continuous molecular evolution, the Liu lab developed the first inhibitory decoy that highly specifically inhibits the constitutively active form of Galpha-q. We use AAV to deliver the gene encoding the evolved decoy, resulting in potent inhibition of uveal melanoma cell proliferation, induction of programmed cell death, and blockade of tumorigenesis in uveal melanoma liver metastasis mouse model. We are applying the same strategy to develop inhibitory decoys highly specific to the active forms of other GTPases, including Galpha-s, Galpha-i, and KRas.
Related Publications:
Dorosheva, O., Zou, J., Charpentier, T., Sondek, J., and Liu, R. “Evolved Inhibitory Decoy Targeting Constitutively Active Gaq Signaling in Uveal Melanoma”, submitted.
rliu@email.unc.edu
ACCEPTING DOCTORAL STUDENTS
The Liu laboratory’s research interests focus on the development and application of novel drug target-binding affinity molecules by integrating directed molecular selection and evolution, ligand design and engineering, in vitro cellular and signaling characterization, and in vivo therapeutic efficacy studies in tumor mouse models. The Liu laboratory has extensive experiences in the design, synthesis, characterization, and delivery of diagnostic and therapeutic agents based on both polypeptides and polynucleotides.
