Optimized serum stability and specificity of an αvß6 integrin-binding peptide for tumor targeting.

The integrin αvß6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvß6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvß6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvß6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvß6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvß6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvß6 in vivo, expanding opportunities for cancer targeting and therapy.

Discovery of a Transferrin Receptor 1-Binding Aptamer and Its Application in Cancer Cell Depletion for Adoptive T-Cell Therapy Manufacturing.

The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novo model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.

Biomaterials in Chimeric Antigen Receptor T-Cell Process Development.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the cancer treatment landscape, utilizing ex vivo modified autologous T cells to treat relapsed or refractory B-cell leukemias and lymphomas. However, the therapy's broader impact has been limited, in part, by a complicated, lengthy, and expensive production process. Accordingly, as CAR T-cell therapies are further advanced to treat other cancers, continual innovation in cell manufacturing will be critical to their successful clinical implementation. In this Account, we describe our research efforts using biomaterials to improve the three fundamental steps in CAR T-cell manufacturing: (1) isolation, (2) activation, and (3) genetic modification.Recognizing that clinical T-cell isolation reagents have high cost and supply constraints, we developed a synthetic DNA aptamer and complementary reversal agent technology that isolates label-free CD8+ T cells with high purity and yield from peripheral blood mononuclear cells. Encouragingly, CAR T cells manufactured from both antibody- and aptamer-isolated T cells were comparable in therapeutic potency. Discovery and design of other T-cell specific aptamers and corresponding reversal reagents could fully realize the potential of this approach, enabling inexpensive isolation of multiple distinct T-cell populations in a single isolation step.Current ex vivo T-cell activation materials do not accurately mimic in situ T-cell activation by antigen presenting cells (APCs). They cause unequal CD4+ and CD8+ T-cell expansion, necessitating separate production of CD4+ and CD8+ CAR T cells for therapies that call for balanced infusion compositions. To address these shortcomings, we designed a panel of biodegradable cell-templated silica microparticles with supported lipid bilayers that display stimulatory ligands for T-cell activation. High membrane fluidity, elongated shape, and rough surface topography, all properties of endogenous APCs, were found to be favorable parameters for activation, promoting unbiased and efficient CD4/CD8 T-cell expansion while not terminally differentiating the cells.Viral and electroporation-based gene delivery systems have various drawbacks. Viral vectors are expensive and have limited cargo sizes, whereas electroporation is highly cytotoxic. Thus, low-cost nonviral platforms that transfect T cells with low cytotoxicity and high efficiency are needed for CAR gene delivery. Our group thus synthesized a panel of cationic polymers with different architectures and evaluated their T-cell transfection ability. We identified a comb-shaped polymer formulation that transfected primary T cells with low cytotoxicity, although transfection efficiency was low compared to conventional methods. Analysis of intracellular and extracellular barriers to transfection revealed low uptake of polyplexes and high endosomal pH in T cells, alluding to biological and polymer properties that could be further improved.These innovations represent just a few recent developments in the biomaterials field for addressing CAR T-cell production needs. Together, these technologies and their future advancement will pave the way for economical and straightforward CAR T-cell manufacturing.

Discovery and Characterization of Spike N-Terminal Domain-Binding Aptamers for Rapid SARS-CoV-2 Detection.

The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×105  copies mL-1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.

anti-EGFR capture mitigates EMT- and chemoresistance-associated heterogeneity in a resistance-profiling CTC platform.

Capture and analysis of circulating tumor cells (CTCs) holds promise for diagnosing and guiding treatment of pancreatic cancer. To accurately monitor disease progression, capture platforms must be robust to processes that increase the phenotypic heterogeneity of CTCs. Most CTC-analysis technologies rely on the recognition of epithelial-specific markers for capture and identification, in particular the epithelial cell-adhesion molecule (EpCAM) and cytokeratin. As the epithelial-to-mesenchymal transition (EMT) and the acquisition of chemoresistance are both associated with loss of epithelial markers and characteristics, the effect of these processes on the expression of commonly used CTC markers, specifically EpCAM, EGFR and cytokeratin, requires further exploration. To determine this effect, we developed an in vitro model of EMT and acquired gemcitabine resistance in human pancreatic cancer cell lines. Using this model, we show that EMT-induction and acquired chemoresistance decrease EpCAM expression and microfluidic anti-EpCAM capture performance. Furthermore, we find that EGFR capture is more robust to these processes. By measuring the expression of known mediators of chemoresistance in captured cells using automated imaging and image processing, we demonstrate the ability to resistance-profile cells on-chip. We expect that this approach will allow for the development of improved non-invasive biomarkers of pancreatic cancer progression.

Aptamer-Based Chromatographic Methods for Efficient and Economical Separation of Leukocyte Populations.

The manufacturing process of chimeric antigen receptor T cell therapies includes isolation systems that provide pure T cells. Current magnetic-activated cell sorting and immunoaffinity chromatography methods produce desired cells with high purity and yield but require expensive equipment and reagents and involve time-consuming incubation steps. Here, we demonstrate that aptamers can be employed in a continuous-flow resin platform for both depletion of monocytes and selection of CD8+ T cells from peripheral blood mononuclear cells at low cost with high purity and throughput. Aptamer-mediated cell selection could potentially enable fully synthetic, traceless isolations of leukocyte subsets from a single isolation system.

Acquired chemoresistance drives spatial heterogeneity, chemoprotection and collective migration in pancreatic tumor spheroids.

Tumors display rich cellular heterogeneity and typically consist of multiple co-existing clones with distinct genotypic and phenotypic characteristics. The acquisition of resistance to chemotherapy has been shown to contribute to the development of aggressive cancer traits, such as increased migration, invasion and stemness. It has been hypothesized that collective cellular behavior and cooperation of cancer cell populations may directly contribute to disease progression and lack of response to treatment. Here we show that the spontaneous emergence of chemoresistance in a cancer cell population exposed to the selective pressure of a chemotherapeutic agent can result in the emergence of collective cell behavior, including cell-sorting, chemoprotection and collective migration. We derived several gemcitabine resistant subclones from the human pancreatic cancer cell line BxPC3 and determined that the observed chemoresistance was driven of a focal amplification of the chr11p15.4 genomic region, resulting in over-expression of the ribonucleotide reductase (RNR) subunit RRM1. Interestingly, these subclones display a rich cell-sorting behavior when cultured as mixed tumor spheroids. Furthermore, we show that chemoresistant cells are able to exert a chemoprotective effect on non-resistant cells in spheroid co-culture, whereas no protective effect is seen in conventional 2D culture. We also demonstrate that the co-culture of resistant and non-resistant cells leads to collective migration where resistant cells enable migration of otherwise non-migratory cells.

Cell-Templated Silica Microparticles with Supported Lipid Bilayers as Artificial Antigen-Presenting Cells for T Cell Activation.

Biomaterial properties that modulate T cell activation, growth, and differentiation are of significant interest in the field of cellular immunotherapy manufacturing. In this work, a new platform technology that allows for the modulation of various activation particle design parameters important for polyclonal T cell activation is presented. Artificial antigen presenting cells (aAPCs) are successfully created using supported lipid bilayers on various cell-templated silica microparticles with defined membrane fluidity and stimulating antibody density. This panel of aAPCs is used to probe the importance of activation particle shape, size, membrane fluidity, and stimulation antibody density on T cell outgrowth and differentiation. All aAPC formulations are able to stimulate T cell growth, and preferentially promote CD8+ T cell growth over CD4+ T cell growth when compared to commercially available pendant antibody-conjugated particles. T cells cultured with HeLa- and red blood cell-templated aAPCs have a less-differentiated and less-exhausted phenotype than those cultured with spherical aAPCs with matched membrane coatings when cultured for 14 days. These results support continued exploration of silica-supported lipid bilayers as an aAPC platform.

Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy.

Chimeric antigen receptor T-cell therapies using defined product compositions require high-purity T-cell isolation systems that, unlike immunomagnetic positive enrichment, are inexpensive and leave no trace on the final cell product. Here, we show that DNA aptamers (generated with a modified cell-SELEX procedure to display low-nanomolar affinity for the T-cell marker CD8) enable the traceless isolation of pure CD8+ T cells at low cost and high yield. Captured CD8+ T cells are released label-free by complementary oligonucleotides that undergo toehold-mediated strand displacement with the aptamer. We also show that chimeric antigen receptor T cells manufactured from these cells are comparable to antibody-isolated chimeric antigen receptor T cells in proliferation, phenotype, effector function and antitumour activity in a mouse model of B-cell lymphoma. By employing multiple aptamers and the corresponding complementary oligonucleotides, aptamer-mediated cell selection could enable the fully synthetic, sequential and traceless isolation of desired lymphocyte subsets from a single system.

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.