Identification of LAG3 high affinity aptamers by HT-SELEX and Conserved Motif Accumulation (CMA)

LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

[1]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[2]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[3]  C. Benoist,et al.  LAG-3 is not responsible for selecting T helper cells in CD4-deficient mice. , 1996, International immunology.

[4]  D. Vignali,et al.  The CD4‐related molecule, LAG‐3 (CD223), regulates the expansion of activated T cells , 2003, European journal of immunology.

[5]  P. Gaulard,et al.  Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand , 2004, Immunogenetics.

[6]  D. Vignali,et al.  Negative Regulation of T Cell Homeostasis by Lymphocyte Activation Gene-3 (CD223)1 , 2005, The Journal of Immunology.

[7]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[8]  K. Jooss,et al.  Lymphocyte Activation Gene-3 Fusion Protein Increases the Potency of a Granulocyte Macrophage Colony-Stimulating Factor–Secreting Tumor Cell Immunotherapy , 2008, Clinical Cancer Research.

[9]  Yoshikazu Nakamura,et al.  Structural and molecular basis for hyperspecificity of RNA aptamer to human immunoglobulin G. , 2008, RNA.

[10]  David H. Mathews,et al.  RNAstructure: software for RNA secondary structure prediction and analysis , 2010, BMC Bioinformatics.

[11]  B. Escudier,et al.  A Phase I Pharmacokinetic and Biological Correlative Study of IMP321, a Novel MHC Class II Agonist, in Patients with Advanced Renal Cell Carcinoma , 2009, Clinical Cancer Research.

[12]  Seung Soo Oh,et al.  Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing , 2010, Proceedings of the National Academy of Sciences.

[13]  T. Haudebourg,et al.  Antibody‐mediated depletion of lymphocyte‐activation gene‐3 (LAG‐3+)‐activated T lymphocytes prevents delayed‐type hypersensitivity in non‐human primates , 2011, Clinical and experimental immunology.

[14]  C. Drake,et al.  Cutting Edge: Accelerated Autoimmune Diabetes in the Absence of LAG-3 , 2011, The Journal of Immunology.

[15]  C. Drake,et al.  LAG-3 in Cancer Immunotherapy. , 2011, Current topics in microbiology and immunology.

[16]  E. Gilboa,et al.  CD28 Aptamers as Powerful Immune Response Modulators , 2013, Molecular therapy. Nucleic acids.

[17]  J. McNamara,et al.  Use of Oligonucleotide Aptamer Ligands to Modulate the Function of Immune Receptors , 2013, Clinical Cancer Research.

[18]  L. Nguyen,et al.  Clinical blockade of PD1 and LAG3 — potential mechanisms of action , 2014, Nature Reviews Immunology.

[19]  S. Inogés,et al.  2-fluoro-RNA oligonucleotide CD40 targeted aptamers for the control of B lymphoma and bone-marrow aplasia. , 2015, Biomaterials.

[20]  K. Odunsi,et al.  LAG3 and PD1 co-inhibitory molecules collaborate to limit CD8+ T cell signaling and dampen antitumor immunity in a murine ovarian cancer model , 2015, Oncotarget.

[21]  Rhiju Das,et al.  Modeling complex RNA tertiary folds with Rosetta. , 2015, Methods in enzymology.

[22]  Khalid K. Alam,et al.  FASTAptamer: A Bioinformatic Toolkit for High-throughput Sequence Analysis of Combinatorial Selections , 2015, Molecular therapy. Nucleic acids.

[23]  J. Lasarte,et al.  Targeting inhibition of Foxp3 by a CD28 2'-Fluro oligonucleotide aptamer conjugated to P60-peptide enhances active cancer immunotherapy. , 2016, Biomaterials.

[24]  Obdulia Rabal,et al.  In Silico Aptamer Docking Studies: From a Retrospective Validation to a Prospective Case Study'TIM3 Aptamers Binding. , 2016, Molecular therapy. Nucleic acids.

[25]  S. Hervás-Stubbs,et al.  Identification of TIM3 2′-fluoro oligonucleotide aptamer by HT-SELEX for cancer immunotherapy , 2015, Oncotarget.

[26]  F. Pastor.  Aptamers: A New Technological Platform in Cancer Immunotherapy , 2016, Pharmaceuticals.

[27]  E. Gilboa,et al.  A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice. , 2017, Molecular therapy : the journal of the American Society of Gene Therapy.

[28]  J. Kolls,et al.  LAG3 limits regulatory T cell proliferation and function in autoimmune diabetes , 2017, Science Immunology.

[29]  J. Rossi,et al.  Aptamers as targeted therapeutics: current potential and challenges , 2016, Nature Reviews Drug Discovery.

[30]  M. Berezovski,et al.  Aptamers for CD Antigens: From Cell Profiling to Activity Modulation , 2016, Molecular therapy. Nucleic acids.