Designing Two-Dimensional Protein Arrays through Fusion of Multimers and Interface Mutations.

We have combined fusion of oligomers with cyclic symmetry and alanine substitutions to eliminate clashes and produce proteins that self-assemble into 2-D arrays upon addition of calcium ions. Using TEM, AFM, small-angle X-ray scattering, and fluorescence microscopy, we show that the designed lattices which are 5 nm high with p3 space group symmetry and 7.25 nm periodicity self-assemble into structures that can exceed 100 µm in characteristic length. The versatile strategy, experimental approach, and hexagonal arrays described herein should prove valuable for the engineering of functional nanostructured materials in 2-D.

Arming Immune Cell Therapeutics with Polymeric Prodrugs.

Engineered immune cells are an exciting therapeutic modality, which survey and attack tumors. Backpacking strategies exploit cell targeting capabilities for delivery of drugs to combat tumors and their immune-suppressive environments. Here, a new platform for arming cell therapeutics through dual receptor and polymeric prodrug engineering is developed. Macrophage and T cell therapeutics are engineered to express a bioorthogonal single chain variable fragment receptor. The receptor binds a fluorescein ligand that directs cell loading with ligand-tagged polymeric prodrugs, termed "drugamers." The fluorescein ligand facilitates stable binding of drugamer to engineered macrophages over 10 days with 80% surface retention. Drugamers also incorporate prodrug monomers of the phosphoinositide-3-kinase inhibitor, PI-103. The extended release of PI-103 from the drugamer sustains antiproliferative activity against a glioblastoma cell line compared to the parent drug. The versatility and modularity of this cell arming system is demonstrated by loading T cells with a second fluorescein-drugamer. This drugamer incorporates a small molecule estrogen analog, CMP8, which stabilizes a degron-tagged transgene to provide temporal regulation of protein activity in engineered T cells. These results demonstrate that this bioorthogonal receptor and drugamer system can be used to arm multiple immune cell classes with both antitumor and transgene-activating small molecule prodrugs.

Preclinical Evaluation of Bispecific Adaptor Molecule Controlled Folate Receptor CAR-T Cell Therapy With Special Focus on Pediatric Malignancies.

Chimeric antigen receptor (CAR)-T cell therapy has transformed pediatric oncology by producing high remission rates and potent effects in CD19+ B-cell malignancies. This scenario is ideal as CD19 expression is homogeneous and human blood provides a favorable environment for CAR-T cells to thrive and destroy cancer cells (along with normal B cells). Yet, CAR-T cell therapies for solid tumors remain challenged by fewer tumor targets and poor CAR-T cell performances in a hostile tumor microenvironment. For acute myeloid leukemia and childhood solid tumors such as osteosarcoma, the primary treatment is systemic chemotherapy that often falls short of expectation especially for relapsed and refractory conditions. We aim to develop a CAR-T adaptor molecule (CAM)-based therapy that uses a bispecific small-molecule ligand EC17, fluorescein isothiocyanate (FITC) conjugated with folic acid, to redirect FITC-specific CAR-T cells against folate receptor (FR)-positive tumors. As previously confirmed in rodents as well as in human clinical studies, EC17 penetrates solid tumors within minutes and is retained due to high affinity for the FR, whereas unbound EC17 rapidly clears from the blood and from receptor-negative tissues. When combined with a rationally designed CAR construct, EC17 CAM was shown to trigger CAR-modified T cell activation and cytolytic activity with a low FR threshold against tumor targets. However, maximal cytolytic potential correlated with (i) functional FR levels (in a semi-log fashion), (ii) the amount of effector cells present, and (iii) tumors' natural sensitivity to T cell mediated killing. In tumor-bearing mice, administration of EC17 CAM was the key to drive CAR-T cell activation, proliferation, and persistence against FR+ pediatric hematologic and solid tumors. In our modeling systems, cytokine release syndrome (CRS) was induced under specific conditions, but the risk of severe CRS could be easily mitigated or prevented by applying intermittent dosing and/or dose-titration strategies for the EC17 CAM. Our approach offers the flexibility of antigen control, prevents T cell exhaustion, and provides additional safety mechanisms including rapid reversal of severe CRS with intravenous sodium fluorescein. In this paper, we summarize the translational aspects of our technology in support of clinical development.

A Self-Assembling Two-Dimensional Protein Array is a Versatile Platform for the Assembly of Multicomponent Nanostructures.

Rationally designed two-dimensional (2D) arrays that support the assembly of nanoscale components are of interest for catalysis, sensing, and biomedical applications. The computational redesign of a protein called TTM that undergoes calcium-induced self-assembly into nanostructured lattices capable of growing to dozens of micrometers are previously reported. The work demonstrates here that the N- and C-termini of the constituent monomers are solvent-accessible and that they can be modified with a hexahistidine extension, a gold-binding peptide, or a biotinylation tag to decorate nickel-nitriloacetic acid beads with self-assembled protein islands, conjugate gold nanoparticles to planar arrays, or control the immobilization density of avidin molecules onto 2D lattices through co-polymerization of biotinylated and wild type TTM monomers. These results showcase the potential of TTM as a versatile 2D scaffold for the fabrication of hierarchical structures comprising a broad range of nanoscale elements.

Self-assembled two-dimensional protein arrays in bionanotechnology: from S-layers to designed lattices.

Although the crystalline S-layer arrays that form the exoskeleton of many archaea and bacteria have been studied for decades, a long-awaited crystal structure coupled with a growing understanding of the S-layer assembly process are injecting new excitement in the field. The trend is amplified by computational strategies that allow for in silico design of protein building blocks capable of self-assembling into 2D lattices and other prescribed quaternary structures. We review these and other recent developments toward achieving unparalleled control over the geometry, chemistry and function of protein-based 2D objects from the nanoscale to the mesoscale.

A genetic approach for controlling the binding and orientation of proteins on nanoparticles.

Although silver nanoparticles are excellent surface enhancers for Raman spectroscopy, their use to probe the conformation of large proteins at interfaces has been complicated by the fact that many polypeptides adsorb weakly or with a random orientation to colloidal silver. To address these limitations, we sought to increase binding affinity and control protein orientation by fusing a silver-binding dodecapeptide termed Ag4 to the C-terminus of maltose-binding protein (MBP), a well-characterized model protein with little intrinsic silver binding affinity. Quartz crystal microbalance measurements conducted with the MBP-Ag4 fusion protein revealed that its affinity for silver (Kd approximately 180 nM) was at least 1 order of magnitude higher than a control protein, MBP2, containing a non-silver-specific C-terminal extension. Under our experimental conditions, MBP-Ag4 SERS spectra exhibited 2-4 fold higher signal-to-background relative to MPB2 and contained a number of amino acid-assigned vibrational modes that were either weak or absent in control experiments performed with MBP2. Changes in amino acid-assigned peaks before and after MBP-Ag4 bound maltose were used to assess protein orientation on the surface of silver nanoparticles. The genetic route described here may prove useful to study the orientation of other proteins on a variety of SERS-active surfaces, to improve biosensors performance, and to control functional nanobiomaterials assembly.