Published in Blood: Characterizing Gene Edits in Allogeneic CAR-T Products with Single-Cell Functional Proteomics

Since the first FDA approval in 2017, chimeric antigen receptor (CAR) T cells, known as CAR-T therapies, have shifted the treatment landscape for patients diagnosed with hematological malignancies. Despite research advancements and subsequent CAR-T approvals in recent years, researchers are still working to refine these therapies to reduce adverse reactions and improve efficacy.

Autologous CAR-T cells are derived directly from the patient’s own cells. Though autologous CAR-T cells reduce the risk of immune rejection, it requires a demanding and lengthy manufacturing process and are specific to one patient. Alternatively, allogeneic, or off-the-shelf, CAR-T products start with cells from healthy donors and are engineered to provide compatible cells on-demand to ease patient burden and decrease treatment delays often associated with autologous therapies. Though promising, this method does come with hurdles, including higher risk of rejection and a lack of approved therapies.

To optimize CAR-T products and decrease the risk of rejection, several different methods have been used. Traditional genome editing techniques use nuclease enzymes that induce double strand breaks (DSBs), which can be unpredictable and result in off target effects. Newer genome editing utilizes cytosine base editors (CBEs), which can create point mutations and inhibit base excision repair, resulting in more precise genome editing. Though this technique is promising, the use of CBEs is still new and must be clinically validated before use in cellular therapies. Single-cell proteomics, previously shown to be a powerful tool for evaluating CAR-T product functionality, can be used to assess the effects of CBEs in cell therapies.

Characterizing Gene-Edited CAR-Ts in the Clinical Setting

In a study recently published in Blood, researchers tested the utility of CBEs to create allogeneic CAR-T products for relapsed/refractory T-cell acute lymphoblastic leukemia (r/r T-ALL). This type of leukemia is difficult to treat as it does not respond well to chemotherapy, and autologous CAR-T products may be already compromised by cancer cells. In this study, the researchers used CBEs to develop a first-of-its-kind quadruple-base-edited allogeneic CAR-T targeted to CD7, a cell surface receptor associated with T-ALL.

Gene Editing to Increase Polyfunctionality

As PD1 signaling is inhibitory and attenuates T cell activity, the authors first explored whether silencing PD1 expression could improve cell function. Using cells from two donors, researchers created 7CAR8, a quadruple-edited CAR-T, with and without PD1 expression and cultured them with recombinant human CD7. They then used IsoPlexis’ Single-Cell Secretome platform to assess the functional phenotypes of the engineered cells. Researchers found that the PD1 knockout increased the polyfunctionality, the ability of a cell to secrete more than one cytokine simultaneously, of 7CAR8 cells in one donor, but not the other. Because polyfunctionality has previously been shown to be associated with clinical outcomes and CAR-T potency, the researchers decided to incorporate the PDCD1 silencing edit into the subsequent 7CAR8 product for in vivo testing. Further experiments demonstrated that the 7CAR8 cells had anti-tumor effects and significantly increased survival in mouse models of T-ALL.

Using Polyfunctionality to Inform CAR-T Design

This study demonstrates how polyfunctionality and single-cell functional phenotyping can help to optimize CAR-T product design. By assessing cell function before clinical trials, researchers gain valuable insights to help strategize development of their early-stage cell therapies. Single-cell analysis with IsoPlexis’ fully automated, end-to-end solutions provides unique insights into CAR-T cell function and potency, helping researchers improve cell therapies and patient outcomes.

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