Activation-inducible CAR expression enables precise control over engineered CAR T cell function.

CAR T cell therapy is a rapidly growing area of oncological treatments having a potential of becoming standard care for multiple indications. Coincidently, CRISPR/Cas gene-editing technology is entering next-generation CAR T cell product manufacturing with the promise of more precise and more controllable cell modification methodology. The intersection of these medical and molecular advancements creates an opportunity for completely new ways of designing engineered cells to help overcome current limitations of cell therapy. In this manuscript we present proof-of-concept data for an engineered feedback loop. We manufactured activation-inducible CAR T cells with the help of CRISPR-mediated targeted integration. This new type of engineered T cells expresses the CAR gene dependent on their activation status. This artifice opens new possibilities to regulate CAR T cell function both in vitro and in vivo. We believe that such a physiological control system can be a powerful addition to the currently available toolbox of next-generation CAR constructs.

A novel polymer-conjugated human IL-15 improves efficacy of CD19-targeted CAR T-cell immunotherapy.

Chimeric antigen receptor (CAR)-modified T-cell therapies targeting CD19 represent a new treatment option for patients with relapsed/refractory (R/R) B-cell malignancies. However, CAR T-cell therapy fails to elicit durable responses in a significant fraction of patients. Limited in vivo proliferation and survival of infused CAR T cells are key causes of failure. In a phase 1/2 clinical trial of CD19 CAR T cells for B-cell malignancies (#NCT01865617), low serum interleukin 15 (IL-15) concentration after CAR T-cell infusion was associated with inferior CAR T-cell kinetics. IL-15 supports T-cell proliferation and survival, and therefore, supplementation with IL-15 may enhance CAR T-cell therapy. However, the clinical use of native IL-15 is challenging because of its unfavorable pharmacokinetic (PK) and toxicity. NKTR-255 is a polymer-conjugated IL-15 that engages the entire IL-15 receptor complex (IL-15Rα/IL-2Rßγ) and exhibits reduced clearance, providing sustained pharmacodynamic (PD) responses. We investigated the PK and immune cell PDs in nonhuman primates treated with NKTR-255 and found that NKTR-255 enhanced the in vivo proliferation of T cells and natural killer cells. In vitro, NKTR-255 induced dose-dependent proliferation and accumulation of human CD19 CAR T cells, especially at low target cell abundance. In vivo studies in lymphoma-bearing immunodeficient mice demonstrated enhanced antitumor efficacy of human CD19 CAR T cells. In contrast to mice treated with CAR T cells alone, those that received CAR T cells and NKTR-255 had markedly higher CAR T-cell counts in the blood and marrow that were sustained after tumor clearance, without evidence of persistent proliferation or ongoing activation/exhaustion as assessed by Ki-67 and inhibitory receptor coexpression. These data support an ongoing phase 1 clinical trial of combined therapy with CD19 CAR T cells and NKTR-255 for R/R B-cell malignancies.

Next generation automated traceless cell chromatography platform for GMP-compliant cell isolation and activation.

Large-scale target cell isolation from patient blood preparations is one of the critical operations during drug product manufacturing for personalized cell therapy in immuno-oncology. Use of high-affinity murine antibody coated magnetic nanoparticles that remain on isolated cells is the current standard applied for this purpose. Here, we present the transformation of previously described technology - non-magnetic immunoaffinity column chromatography-based cell selection with reversible reagents into a new clinical-grade cell isolation platform called Automated Traceless Cell affinity chromatography (ATC). ATC is a fully closed and GMP-compliant cell selection and manufacturing system. Reversibility of reagents enables (sequential) positive cell selection, optionally in combination with depletion columns, enabling capture of highly specific cell subsets. Moreover, synergy with other Streptamer-based technologies allows novel uses beyond cell isolation including integrated and automated on-column target cell activation. In conclusion, ATC technology is an innovative as well as versatile platform to select, stimulate and modify cells for clinical manufacturing and downstream therapies.

Expamers: a new technology to control T cell activation.

T cell activation is a cornerstone in manufacturing of T cell-based therapies, and precise control over T cell activation is important in the development of the next generation T-cell based therapeutics. This need cannot be fulfilled by currently available methods for T cell stimulation, in particular not in a time dependent manner. Here, we describe a modular activation reagent called Expamers, which addresses these limitations. Expamers are versatile stimuli that are intended for research and clinical use. They are readily soluble and can be rapidly bound and removed from the cell surface, allowing nearly instantaneous initiation and termination of activation signal, respectively. Hence, Expamers enable precise regulation of T cell stimulation duration and provide promise of control over T cell profiles in future products. Expamers can be easily adopted to different T cell production formats and have the potential to increase efficacy of T cell immunotherapeutics.