Development of a high-throughput pipeline for discovery of T cell based immunotherapeutics

Redirecting T cells towards tumor-associated antigens (TAAs) is an emerging treatment modality in cancer immunotherapy. Two key redirection strategies widely used, involve engineering exogenous receptors on T cells termed chimeric antigen receptors (CARs) and the usage of soluble bispecific antibodies called Bispecific T cell engagers (BiTEs) to ligate T cells with cancer cells expressing specific TAAs. Both BiTE and CAR-T based treatment strategies have been clinically approved for CD19 positive B cell-based cancers. However, this success has not been clinically translatable to other targets. A key challenge to the limited success of T cell redirection therapies across differ cancer types is the non-availability of a versatile discovery platform that facilitates quick functional screening. In this work, we describe the development of a novel high throughput discovery pipeline for T cell-based redirection therapies. In particular, we utilize a droplet based microfluidic system to assess a predefined library of BiTE variants and report the discovery of novel functional BiTE sequences. We also obtain insights into hitherto undiscovered BiTE design principles which could inform future discovery. In addition, we also present a proof-of-concept study to adapt the droplet microfluidic based platform for discovery of CAR-Ts

CAR-T design: Elements and their synergistic function.

Chimeric antigen receptor (CAR) T cells use re-engineered cell surface receptors to specifically bind to and lyse oncogenic cells. Two clinically approved CAR-T-cell therapies have significant clinical efficacy in treating CD19-positive B cell cancers. With widespread interest to deploy this immunotherapy to other cancers, there has been great research activity to design new CAR structures to increase the range of targeted cancers and anti-tumor efficacy. However, several obstacles must be addressed before CAR-T-cell therapies can be more widely deployed. These include limiting the frequency of lethal cytokine storms, enhancing T-cell persistence and signaling, and improving target antigen specificity. We provide a comprehensive review of recent research on CAR design and systematically evaluate design aspects of the four major modules of CAR structure: the ligand-binding, spacer, transmembrane, and cytoplasmic domains, elucidating design strategies and principles to guide future immunotherapeutic discovery

CAR-T design: Elements and their synergistic function.

Chimeric antigen receptor (CAR) T cells use re-engineered cell surface receptors to specifically bind to and lyse oncogenic cells. Two clinically approved CAR-T-cell therapies have significant clinical efficacy in treating CD19-positive B cell cancers. With widespread interest to deploy this immunotherapy to other cancers, there has been great research activity to design new CAR structures to increase the range of targeted cancers and anti-tumor efficacy. However, several obstacles must be addressed before CAR-T-cell therapies can be more widely deployed. These include limiting the frequency of lethal cytokine storms, enhancing T-cell persistence and signaling, and improving target antigen specificity. We provide a comprehensive review of recent research on CAR design and systematically evaluate design aspects of the four major modules of CAR structure: the ligand-binding, spacer, transmembrane, and cytoplasmic domains, elucidating design strategies and principles to guide future immunotherapeutic discovery

A high throughput bispecific antibody discovery pipeline

A single-cell based bispecific antibody (BsAb) discovery pipeline, based on a microfluidics droplets generation and sorting systems, has the potential to speed up the discovery and development of functional antibodies such as BsAb therapeutics

A high throughput bispecific antibody discovery pipeline

Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy, but inefficiency in the current discovery has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model, we demonstrate that our single-cell platform possesses a high throughput screening efficiency of up to one and a half million variant library cells per run and can isolate rare functional clones at a low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones, including extremely rare ones (~ 0.001% abundance). We also discovered BiTEs that exhibit novel properties and insights to design variable preferences for functionality. We expect our single-cell platform to not only increase the discovery efficiency of new immunotherapeutics, but also enable identifying generalizable design principles based on an in-depth understanding of the inter-relationships between sequence, structure, and function

Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.

To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders