Implantable CAR T cell factories enhance solid tumor treatment.

Chimeric Antigen Receptor (CAR) T cell therapy has produced revolutionary success in hematological cancers such as leukemia and lymphoma. Nonetheless, its translation to solid tumors faces challenges due to manufacturing complexities, short-lived in vivo persistence, and transient therapeutic impact. We introduce 'Drydux' - an innovative macroporous biomaterial scaffold designed for rapid, efficient in-situ generation of tumor-specific CAR T cells. Drydux expedites CAR T cell preparation with a mere three-day turnaround from patient blood collection, presenting a cost-effective, streamlined alternative to conventional methodologies. Notably, Drydux-enabled CAR T cells provide prolonged in vivo release, functionality, and enhanced persistence exceeding 150 days, with cells transitioning to memory phenotypes. Unlike conventional CAR T cell therapy, which offered only temporary tumor control, equivalent Drydux cell doses induced lasting tumor remission in various animal tumor models, including systemic lymphoma, peritoneal ovarian cancer, metastatic lung cancer, and orthotopic pancreatic cancer. Drydux's approach holds promise in revolutionizing solid tumor CAR T cell therapy by delivering durable, rapid, and cost-effective treatments and broadening patient accessibility to this groundbreaking therapy.

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells.

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce >85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.

Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells.

Despite their clinical success, chimeric antigen receptor (CAR)-T cell therapies for B cell malignancies are limited by lengthy, costly and labor-intensive ex vivo manufacturing procedures that might lead to cell products with heterogeneous composition. Here we describe an implantable Multifunctional Alginate Scaffold for T Cell Engineering and Release (MASTER) that streamlines in vivo CAR-T cell manufacturing and reduces processing time to a single day. When seeded with human peripheral blood mononuclear cells and CD19-encoding retroviral particles, MASTER provides the appropriate interface for viral vector-mediated gene transfer and, after subcutaneous implantation, mediates the release of functional CAR-T cells in mice. We further demonstrate that in vivo-generated CAR-T cells enter the bloodstream and control distal tumor growth in a mouse xenograft model of lymphoma, showing greater persistence than conventional CAR-T cells. MASTER promises to transform CAR-T cell therapy by fast-tracking manufacture and potentially reducing the complexity and resources needed for provision of this type of therapy.

Scaffold-Mediated Static Transduction of T Cells for CAR-T Cell Therapy.

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive clinical responses in patients with B-cell malignancies. Critical to the success of CAR-T cell therapies is the achievement of robust gene transfer into T cells mediated by viral vectors such as gamma-retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost-effective CAR-T cell therapies. Herein, a low-cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition is described. CAR-T cells produced by macroporous scaffold-mediated viral transduction exhibit >60% CAR expression, retain effector phenotype, expand to clinically relevant cell numbers, and eradicate CD19+ lymphoma in vivo. Efficient transduction is dependent on scaffold macroporosity. Taken together, the data show that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.

Glucocorticoid receptor-mediated delivery of nano gold-withaferin conjugates for reversal of epithelial-to-mesenchymal transition and tumor regression.

AIM: To explore the potential of glucocorticoid receptor-targeted nano-gold formulation as antitumor drug sensitizing agent. MATERIALS & METHODS: Simultaneous conjugation of gold nanoparticle with thiol-modified dexamethasone, a synthetic glucocorticoid and anticancer drug withaferin A afforded stable gold nanoparticle-modifed dexamethasone-withaferin A nanoconjugate. RESULTS: This metallic nanoparticle formulation showed glucocorticoid receptor-dependent cancer cell selective cytotoxicity, inhibited growth of aggressive mouse melanoma tumor, reduced mice mortality, while reversing epithelial-to-mesenchymal transition in tumor cells. Same treatment also leads to near-complete downregulation of ABCG2 drug transporter in tumor-associated cells thus attributing it to its drug sensitizing ability. CONCLUSION: The presently synthesized nanoconjugate holds a great promise to sensitize cancer cells to chemotherapeutics and induce epithelial-to-mesenchymal transition reversal in tumor cells preventing metastasis.

N-end rule pathway inhibition assists colon tumor regression via necroptosis.

Recent study has shown that N-end rule pathway, an ubiquitin dependent proteolytic system, counteracts cell death by degrading many antisurvival protein fragments like BCLxL, BRCA1, RIPK1, etc. Inhibition of the N-end rule pathway can lead to metabolic stabilization of proapoptotic protein fragments like RIPK1, thereby sensitizing cells to programmed cell death. Receptor interacting serine-threonine protein kinase-1 (RIPK1) is one of the upstream regulators of programmed necrosis known as necroptosis. Necroptosis is particularly gaining attention of cancer biologists as it provides an alternate therapeutic modality to kill cancer cells, which often evolve multiple strategies to circumvent growth inhibition by apoptosis. Utilizing the over expression of biotin receptor in cancer cells, herein, we report that coadministration of synthetic hetero-bivalent N-end rule inhibitor RFC11 and anticancer drug shikonin solubilized in a stable biotin receptor-targeted liposome exhibited significant synergistic antitumor effect in both subcutaneous and orthotopic mouse colon tumor model through induction of necroptosis with distinctive upregulation of RIPK1. Besides developing a newly targeted formulation for necroptosis induction, this report is the first in vivo evidence demonstrating that potent inhibition of N-end rule pathway can enhance therapeutic efficacy of conventional chemotherapeutics.

Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle.

Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of 'exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of 'endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of 'endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.