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January 16, 2018

3D Bioscaffold Mimics Antigen-Presenting Cells for Improved T-Cell Expansion

The left image shows a scanning electron micrograph (SEM) of a basic scaffold made of many tiny mesoporous silica rods (MSRs) before they are coated with a thin supported lipid bilayer (SLB) with the incorporated T cell-stimulating cues. In the right SEM image, T cells (in blue) bind to a section of a completed antigen-presenting cell-mimetic scaffold (in brown), where they are instructed to multiply and are kept alive for future use in T cell therapies. [Wyss Institute at Harvard University]

  • Researchers in the U.S. have developed a 3D biomaterial scaffold that can promote much faster ex vivo expansion of functional T cells than currently available technologies. The new scaffold is designed to provide the cues that are delivered to T cells by the body’s own antigen-presenting cells (APCs), in context, and so better mimic in vivo signaling by APCs. Led by David Mooney, Ph.D., at Harvard's Wyss Institute for Biologically Inspired Engineering and John A. Paulson School of Engineering and Applied Sciences (SEAS), the team demonstrated how the technology dramatically increased the ex vivo expansion of both primary mouse and human T cells, as well as of cancer-targeting chimeric antigen receptor (CAR) T cells, which they successfully tested in a mouse model of lymphoma. The researchers project that the technology could broaden the utility of immunotherapeutics, including adoptive cell transfer (ACT). 

    “Our approach closely mimics how APCs present their stimulating cues to primary T cells on their outer membrane and how they release soluble factors that enhance the survival of the T cells,” comments Dr. Mooney, who is a core faculty member at the Wyss Institute, leader of its Immunomaterials Platform, and also the Robert P. Pinkas Family Professor of Bioengineering at SEAS. “As a result, we achieve much faster and greater expansion. By varying the compositions of lipids, cues, and diffusible factors in the scaffolds, we engineered a very versatile and flexible platform that can be used to amplify specific T-cell populations from blood samples, and that could be deployed in existing therapies such as CAR-T therapies.”

    The team reports on the technology in Nature Biotechnology, in a paper entitled “Scaffolds That Mimic Antigen-Presenting Cells Enable Ex Vivo Expansion of Primary T Cells.”

    T-cell-based therapies represent a “promising approach” for treating a variety of diseases and have already shown shown “unprecedented” clinical success in treating B-cell acute lymphoblastic leukemia and non-Hodgkin’s lymphoma, the researchers write. Unfortunately, promoting the rapid ex vivo expansion of T cells for ACT therapies, a key step for adoptive cell transfer techniques, “remains a challenge.”

    T-cell activation requires three signals: T-cell receptor (TCR) stimulation, costimulation, and prosurvival cytokines. In vivo, these signaling cues are presented to T cells by APCs, but triggering the expansion of T cells ex vivo is trickier. Synthetic artificial APCs, such as Dynabeads, are used to expand T cells for clinical applications. But while Dynabeads provide the T cells with the right signals, the cues are not necessarily presented in the same context that they would be presented in vivo, the authors explain. This can result in less than optimal T-cell expansion and functionality. Another common system for triggering antigen-specific expansion of naïve and memory T cells is the use of autologous monocyte-derived dendritic cells (moDCs), but this approach has drawbacks, including lengthy manufacturing processes and variability among donor moDCs.

    As an alternative approach, Dr. Mooney’s team developed a composite scaffold material that is designed to mimic APC signaling to promote the expansion of T cells ex vivo. To construct the APC-mimetic scaffold (APC-ms), the researchers first loaded mesoporous silica rods (MSRs) with interleukin 2 (IL-2), a factor produced by APCs that prolongs the survival of T cells. The MSRs were coated with lipids that formed a thin supported lipid bilayer (SLB), resembling the outer membrane of APCs. Two T-cell-stimulating antibodies, anti-CD3 and anti-CD28, were added to the SLB bilayer, where they were able to move within the bilayer and bind to receptor/coreceptor molecules on the surface of T cells. When constructed in culture medium, 3D scaffolds spontaneously formed through the settling and random stacking of the rods, forming pores that allow the entry, movement, and accumulation of T cells and signaling them to multiply.

    The team then compared how well different formulations of their APC-ms were able to promote T-cell expansion, in comparison with commercially available Dynabeads. When tested with primary mouse T cells, “culture with the various APC-ms formulations yielded three- to fivefold greater T-cell expansion than all Dynabead conditions tested, with expansion efficiency dependent on the particular APC-ms formulation,” the authors write. Tests to expand primary human cells also showed that, “Culture with all the tested APC-s formulations led to a two- to tenfold greater expansion than with Dynabeads, with >95% of cells viable at 14d.”

    Encouragingly, the 3D APC-ms scaffolds could be designed to promote antigen-specific expansion of primary mouse T cells and supported antigen-specific enrichment and expansion of rare human T-cell subpopulations. This capability could allow the selective expansion of cancer antigen-specific T cells from tumors or blood, the authors suggest. "In a single dose, APC-mimetic scaffolds led to two- to ten-fold greater expansion of primary mouse and human T cells than Dynabeads," notes co-author David Zhang, a graduate student working with Mooney. “As another advantage, APC-mimetic scaffolds enabled us to tune the ratios of subpopulations of T cells with different roles in the desired immune responses, which in the future might increase their functionality."

    Comparative experiments also showed that formulations of the new scaffold could be used to expand specific T-cell populations directly from heterogeneous cell mixtures, including peripheral blood mononuclear cells (PBMCs), and so remove the need to isolate the T cells before expansion, they note. “Taken together, these data demonstrate the ability of APC-ms to robustly expand functional human T cells in an antigen-specific manner from either purified CD8+ T cells or heterogeneous cell populations such as PBMCs,” the team writes.

    "Based also on studies in which we showed that APC-mimetic scaffolds also have superior potential to specifically enrich and expand rare T-cell subpopulations from blood, we strongly believe that we created an effective platform technology that could facilitate more effective precision immunotherapies," says first author Alexander Cheung, Ph.D., who worked with Mooney and is currently at UNUM Therapeutics in Cambridge, Massachusetts.

    In a final set of experiments, the team demonstrated that an APC-mimetic scaffold engineered to activate a specific type of CAR T cell was able to generate higher numbers of the modified T cells over longer periods of culture than analogously designed expansion beads. The resulting cells were similarly effective in killing the lymphoma cells in a mouse model. The ability to use an APC-ms platform for clinical CAR T-cell expansion applications would be particularly beneficial, the authors point out, as it negates the need to extract the Dynabeads from the T-cell population. “…processes for CAR T-cell production that involve the use of Dynabeads require that beads be removed before administration,” the team explains. “Although simple in theory, this can be challenging and inconsistent in practice.”

    “Current synthetic aAPC systems emphasize TCR clustering and subsequent T-cell activation via the static, high-density presentation of stimuli,” the authors continue. “Here we show that by presenting T-cell stimuli across the surface of a fluid lipid bilayer, emulating how these cues are presented on an APC plasma membrane, relatively lower surface cue densities can promote more rapid expansion rates and generate T cells with a more functional and less exhausted phenotype.…APC-ms represents a multifunctional material platform that promotes more efficient polyclonal and antigen-specific cell expansion than widely used T-cell expansion systems (e.g., Dynabeads and moDCs).…We envision that future iterations of APC-ms could present larger sets of both surface and soluble cues, enabling the generation of further optimized T cells for ACT.”

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