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334 CAR T engagers encoding immune checkpoint inhibitors and immune-relevant cytokines stimulate CAR T cell activity against hematologic and solid tumors
  1. Paul Rennert,
  2. Lan Wu,
  3. Lihe Su,
  4. Roy Lobb and
  5. Christine Ambrose
  1. Aleta Biotherapeutics, Natick, MA, USA


Background Cell therapeutics are limited by critical issues including antigen escape, antigen heterogeneity, tumor and tumor microenvironment mediated immune suppression and suboptimal T cell expansion, fitness and persistence. We originally created CAR T Engagers (CTEs) to address the issues of antigen escape and heterogeneity. Here we present novel domains added to CTEs to overcome immune suppression and enhance CAR T cell function.

Methods Our first generation of functionally enhanced CTEs are built on multi-antigen targeting modules. ALETA-001 is a biologic CTE that binds to CD20 and displays the CD19 extracellular domain (ECD). ALETA-002 is a lentiviral construct that expresses an anti-CD19 CAR domain and a CTE that binds both Her2 and B7-H3. ALETA-001 and -002 CTE protein sequences were further modified to contain additional functional domains including an anti-PD-L1 VHH, a monomeric TGFbetaR2 TRAP, the CD2-binding domain of LFA3, a T cell stimulating cytokine, and an immune system activating cytokine. CTEs that were functionally enhanced (FE) were evaluated for their ability to promote anti-CD19 CAR T-mediated cytotoxicity, to overcome immune suppression and immune escape, and to productively stimulate CAR T cells and engage with endogenous immune cells.

Results Functionally enhanced CTE’s directed anti-CD19 CAR T cells to attack and kill cancer cells including B cell lymphomas (ALETA-001-FE) and solid tumors (ALETA-002-FE). Further, ALETA-001-FE-1 countered loss of CD19 antigen and provided additional costimulation via LFA3/CD2 engagement. ALETA-001-FE-2 stimulated CAR T expansion via enhanced engagement of common-gamma chain signaling. ALETA-002-FE-1 and ALETA-002-FE-2 mediated potent killing of Her+, B7H3+ and dual+ solid tumor cells. ALETA-002-FE-1 also blocked TGFb signaling with a sub-nM IC50. ALETA-002-FE-2 blocked PD-L1 activity with similar potency.

Conclusions Anti-CD19 CAR T cells demonstrate best-in-class expansion, fitness and persistence due to their ability to interact productively with normal CD19+ B cells in circulation and within secondary lymphoid organs that provide T cell nurturing signals. Anti-CD19 CAR T cell use for B cell lymphoma therapy was enhanced by limiting antigen escape and increasing costimulation via LFA3/CD2 binding Anti-CD19 CAR T cells can be redirected to solid tumor antigens by the use of CAR T-secreted CTEs. Here we used multi-antigen targeting, ie. by linking the CD19 ECD to anti-Her2 and anti-B7H3 domains, by providing productive cytokine signaling and by countering immune suppression. Notably, all these functions are incorporated into the CTE, leaving CAR T engineering as separate tool. CTEs carrying multiple domains designed to simultaneously enhance functionality are being designed and evaluated and will be presented.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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