New Study Describes Strep-tag Isolation of more efficient CAR T Cells

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Last week we wrote about immunotherapy, and this week we dig a little deeper into one of the many numbers of recent studies that describe new approaches that address improving various aspects of its therapeutic cycle.

Having sufficient numbers of potent cells is a common requirement for T cell-based therapies for a number of indications. To address issues with obtaining, or isolating, large numbers of cells with high specificity to the target therapy, a group of scientists from the Fred Hutchinson Cancer Research Center and the University of Washington Department of Medicine looked at a new way of achieving this by using affinity-specific tags.

Dr. Stanley Riddell and colleagues developed a method to append Strep-tags to different CARs in order to achieve more targeted isolation of cancer-specific engineered T cells.

The Strep-tag is a sequence consisting of the amino acids Trp-Ser-His-Pro-Gln-Phe-Glu-Lys that allows from the rapid isolation of tagged proteins. By adding Strep-tags to CAR proteins, engineered T cells can be isolated by specific affinity-based interaction, which results in highly selective yields. These tags can also be added to natural T-cell receptors.

The study, titled Inclusion of Strep-tag II in design of antigen receptors for T-cell immunotherapy, was published in the journal Nature Biotechnology. By using this approach, the authors found that this technology resulted in a nearly 95 percent pure collection of CAR T cells.

The authors estimate that, by using this approach, cell process times can be cut by up to 50% or more, significant for therapeutic applications.

Two of the authors, Lingfeng Liu and Stanley Riddell patented this technology under “Tagged chimeric effector molecules and receptors thereof.” It was licensed exclusively to Juno Therapeutics.

 

nbt.3461-F1
Representative data of CAR T isolation and functional assays. The caption was adapted from the paper. (a) Analysis of CD19 CAR expression. Nontransduced cells are depicted in white. (b) Cytolytic activity of CD19-Hi and Strep-tag II 4-1BB/CD3ζ CAR-T cells based on lysis of K562/CD19 or K562/ROR1 at various effector/target (E:T) ratios. (c) IFN-γ and IL-2 production by CD19-Hi and Strep-tag 4-1BB/CD3ζ CAR-T cells 24 h after stimulation with K562/CD19 and K562/ROR1. (d) Tumor progression and distribution evaluated by serial bioluminescence imaging in cohorts of NSG mice inoculated with Raji-ffluc and then CD19 4-1BB/CD3ζ CAR-T cells with Hi, 1ST or 3ST spacers. (e) Tracking CAR-expressing T cells in vivo by staining with anti-Strep-tag II mAb. (f) Kinetics of expansion and contraction of CD19 CAR-T cells in the blood after adoptive transfer to NSG mice bearing Raji tumors. (g) Fold-change in expression of selected cytokine genes in CD19-1ST/4-1BB/-CD3ζ CAR-T cells after infusion to Raji tumor-bearing and nontumor-bearing NSG mice and staining with anti-EGFR or anti-Strep-tag mAb. (h) Cytokine production by CD8+ T cells expressing CD19-1ST/4-1BB/CD3ζ CARs after stimulation with Raji cells in vitro.

 

The study is heavily preliminary and, beyond initial mouse studies (see panels d and f in image above for representative data), further in vivo studies are required.

The paradigm addressed by this work is one that has important implication for the ways in which T cells are obtained and isolated, though considerations on the how this treatments falls within the immunotherapeutic workflow still hold. These cells still need to be expanded in culture and conditions must be such that this very aspect is maximized in order to reap most benefit from this technology.

You can read the study here.

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