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228 Engineered human IL-2/IL-2Rß orthogonal pairs selectively enhance anti-GPC3 CAR T cells in vivo to drive complete responses in solid epithelial tumor models
  1. Paul-Joseph Aspuria,
  2. Marie Semana,
  3. Ivan Cheng,
  4. Navneet Ratti,
  5. Mahalaksmi Ramadass,
  6. Deepti Rokkam,
  7. Mohammed Ali,
  8. Michele Bauer,
  9. Ryan Burgess,
  10. Henry Rosas,
  11. Sandro Vivona,
  12. George Zheng,
  13. Patrick Lupardus and
  14. Martin Oft
  1. Synthekine, Menlo Park, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background CAR T cell therapy has demonstrated clinical efficacy against hematological malignancies, while prominent barriers including poor T cell effector function, lack of proliferation, and limited CAR T cell persistence have prevented CAR T cell therapies from reaching their full curative potential, especially in solid tumors. For example, CAR T cell therapies targeting GPC3, whose overexpression is associated with various malignancies such as hepatocellular carcinoma (HCC), have demonstrated modest depths of response.

Interleukin-2 (IL-2) is a potent stimulator of T cell proliferation, survival, and cytotoxic function. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations. To facilitate selective ex vivo and in vivo expansion/activation of engineered T cells, we developed a human orthogonal ligand/receptor system consisting of a half-life extended IL-2 mutein (STK-009) that does not significantly stimulate cells expressing wild type IL-2 receptor and a mutated IL-2 Receptor Beta (hoRb) that responds to STK-009 but not wild type IL-2. This system enables selective in vivo IL-2 signaling in engineered cells that express the hoRb while avoiding stimulation of native IL-2 responsive cells. Previously, we demonstrated the ability of the STK-009/hoRb pair to selectively enhance the anti-tumor efficacy of hoRb expressing CD19 CAR T cells (SYNCAR-001) in preclinical lymphoma mouse models.1 SYNCAR-001 is currently being tested in a clinical trial targeting CD19+ B-cell malignancies (NCT05665062).

Methods We incorporated the hoRb downstream of a second generation anti-GPC3 CAR via a T2A cleavage peptide to allow for bicistronic expression from one lentiviral construct in transduced T cells (SYNCAR-002). SYNCAR-002 was tested in combination with or without STK-009 in various subcutaneous and intraperitoneal mouse models of HCC for anti-tumor response with transcriptomic and immunophenotyping analyses.

Results In vitro, SYNCAR-002 transduced cells could be selectively enriched with STK-009 with a less exhaustive phenotype compared to WT IL-2. In vivo, STK-009 administration enabled tumor rejections of highly aggressive HCC models by SYNCAR-002 and durability of response against tumor rechallenges (figures 1 and 2). Additionally, STK-009 treatment resulted in significant expansion of SYNCAR-002 in the peripheral blood and intratumorally. Importantly, STK-009 induced the expression of cytotoxic machinery, pro-survival, and proliferative genes in tumor-infiltrating SYNCAR-002 (figure 3).

Conclusions These findings validate that the orthogonal IL-2 platform has the potential to improve the efficacy and durability of CAR T therapy for solid tumor targets such as GPC3 by selectively expanding CAR-T cells in vivo and activating CAR-T cells in a hostile tumor microenvironment.


  1. Aspuria, et al. An Orthogonal IL-2 and IL-2Rß System Drives Persistence and Activation of CAR T cells and Bulky Lymphoma. Science Translational Medicine. Dec 2021;13(625).

Abstract 228 Figure 1

SYNCAR-002 + STK-009 control hepatocellular carcinoma tumors with varying degrees of GPC3 expression. SYNCAR-002 -/+ STK-009 efficacy in subcutaneous and intraperitoneal HCC xenograft mouse models. Subcutaneous and intraperitoneal models were treated by a suboptimal dose of SYNCAR-002 T cells with or without subcutaneous injection of STK-009 at <100mm3 and on day 7, respectively. Median tumor volume and tumor burden is displayed.

Abstract 228 Figure 2

Mice previouly cured by SYNCAR-002 + STK-009 withstand HEPG2 tumor rechallenge. HEPG2 rechallenge model. Mice with subcutaneous HEPG2 tumors that received SYNCAR-002 at Day 7 and subsequent doses of STK-009 were cured by Day 50. HEPG2 + Matrigel were implanted into these mice at Day 55 and into naïve mice. Previously cured mice were split into two groups receiving either PBS (n=4) or STK-009 2x/week (n=5).

Abstract 228 Figure 3

STK-009 enhances proliferation and effector activity in intratumoral SYNCAR-002 cells as determined by flow cytometry and GeoMX transcriptomic analysis.

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