Standardized in-vitro evaluation of CAR-T cells using acellular artificial target particles

Abstract

The horizon of immunotherapy using CAR-T cells is continuously extending to treat solid tumors beyond the success in the treatment of liquid tumors. Precise in-vitro evaluations of CAR-T cells for their phenotypes, quantity and quality of activation in various tumor microenvironments including different antigen densities, and the resulting effector functions are critical for the successful development of CAR-T therapies and safe translation to clinics. Unfortunately, the development of methods and tools to accommodate these needs have been lagging behind. Here, we developed a novel biomaterial platform, acellular artificial target particles (aaTPs) against CAR-T cells, using magnetic microbeads that are already widely employed in the manufacturing of T cell products. By devising a simple and standardized procedure, we precisely controlled the antigen surface densities presented on the aaTPs for a wide range. By co-incubation of aaTPs with CAR-T cells followed by flow cytometry and cytokine assays, we quantitatively determined the antigen-specific and dose-dependent activation of anti-HER2 CAR-T cells. We also demonstrated that the aaTP can serve as a clean target cell in in-vitro assays to prove the proposed mechanism of action of a next-generation CAR-T product. Overall, the simple, inexpensive, modular and precisely controllable synthetic nature of aaTPs enables the development of clean and standardized in-vitro assays for CAR-T cells, which provides critical advantages over the conventional assays using target cell lines. The design of aaTPs can be extended to include other tumor antigens and relevant surface molecules of physiological target cells. Thus, the aaTP platform has great potential as a standardized tool for the development and evaluation of both conventional and new CAR-T products in the context of approval from regulatory agencies and clinical translation.

Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

“…To induce IL-12 secretion, we stimulated for 3 days RB-312 and cRB-312 with HER2-coated beads on a 1:1 bead-to-cell ratio in T cell basal media (TBM) as previously described [ 26 ]. As control we used bovine serum albumin (BSA)-coated beads.…”

“…Beads were prepared as previously reported [13,26]. Approximately 80,000 CAR-T cells were cultured in 96-well flat bottom plates and HER2 beads were added at a 1:1 bead to HER2 CAR-T cells ratio, in TBM media.…”

Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro

“…Acellular systems have been previously developed and proven useful to trigger antigen-specific degranulation and cytokine secretion as a measure for CAR T cell activation. Systems based on planar substrates, e.g., protein-functionalized 96-well plates or micropatterned surfaces, or using mobile bead-based platforms that resemble more closely cells have not yet explored the influence of costimulatory factors during CAR T cell activation (21)(22)(23). Other systems even tried to mimic the fluidity of cell membranes by covering a glass surface with a lipid bilayer (41) However, none of these platforms have demonstrated whether the induced CAR T cell activities were comparable to biologically relevant responses directed to B cell malignancies.…”

“…A micropatterned antigen presenting surface with anti-idiotype antibodies and recombinant proteins of adhesion molecules has been reported to induce early activation signals and immunological synapse formation of CAR T cells (22). In addition, recombinant target proteins immobilized on surface plates (23) or on magnetic beads (24) could trigger antigen-dependent CAR T cell degranulation and cytokine secretion. These findings provided insights on the design of acellular target for CAR T cells.…”

Nanomaterials Boost CAR‐T Therapy for Solid Tumors

“…[84,85] To overcome these limitations, Roh's team enhanced T cell viability by incorporating a molecular modification of HER2 onto the surface of iron oxide particles (Dynabeads), creating a cell-free artificial target particle (aaTP) that induces CAR-T cell activation (Figure 3a). [86] In another approach, Weber's team designed a novel artificial antigen-presenting cell (nano-APC) based on paramagnetic iron-dextran nanoparticles coupled with 𝛼CD28 and MHC-Ig molecules loaded with HLA-A2-restricted peptides to stimulate the expansion of human memory T cells and naive tumor-specific T cells, imparting CAR-T cell therapy with efficacy and durability (Figure 3a). [87]…”