MyD88/CD40 Enhanced CD19-Specific CAR-T Cells Maintain Therapeutic Efficacy Following Resolution of Cytokine-Related Toxicity Using Inducible Caspase-9

Introduction: The efficacy of chimeric antigen receptor-modified T cells (CAR-T) targeting CD19⁺ leukemias and lymphomas is dependent on their in vivo expansion following adoptive transfer. Additional genetic augmentations to improve CAR-T expansion may improve therapeutic efficacy but risks increasing CAR-T toxicity. Here, we demonstrate that a highly active CD19-specific CAR-T cell constitutively expressing a MyD88/CD40 (MC) costimulatory fusion protein and the inducible caspase-9 (iC9) safety switch, is effective at eliminating tumors but induces acute cytokine-related toxicity in animal models. This toxicity, however, can be resolved by administration of rimiducid to induce partial CAR-T apoptosis preserving long-term anti-tumor effects.

Methods: T cells were activated with anti-CD3/CD28 antibodies and transduced with a retrovirus encoding iC9, a first-generation CAR (with CD3ζ) targeting CD19, and a constitutively active MC costimulatory protein (SFG-iC9-CD19.ζ-MC). In vitro coculture assays were used to measure specific killing of CD19⁺ tumor cells (Daudi and Raji) and cytokine production. To assess anti-tumor efficacy, immunodeficient NSG mice were injected i.v. with 5x10⁵ Raji-EGFPluciferase (EGFPluc) or 1x10⁶ Daudi-EGFPluc tumor cells followed by i.v. administration of variable doses (1x10⁶ to 1x10⁷) non-transduced (NT) or iC9-CD19.ζ-MC-modified T cells on days 4 to 10 after tumor engraftment. Tumor growth was measured by in vivo imaging. CAR-T cell toxicity was assessed by weight loss and physical appearance. In initial experiments, CAR-T toxicity was resolved by i.p. administration of 0.5 mg/kg rimiducid after 15% weight loss. Serum cytokines were measured before and after rimiducid treatment. In addition, mice were treated with neutralizing antibodies to hIL-6, hIFN-γ and hTNF-α (100 ug i.p. twice weekly). Further, iC9-CD19.ζ-MC-modified T cells were sorted into CD4⁺ and CD8⁺ T cell fractions to assess the impact of cytokine production from CAR-T cells on animal health. Mice treated with iC9-CD19.ζ-MC-modified T cells that also received rimiducid to resolve toxicity were subsequently rechallenged with tumors to measure persistence of residual CAR-T cells, and at the end of the experiment were sacrificed and the spleens analyzed for the presence of human T cells and functional CAR-T activity using in vitro assays.

Results: T cells were efficiently transduced (40-85%) with the iC9-CD19.ζ-MC retrovirus. In vitro assays showed that these CAR-T cells eliminated CD19+ Raji and Daudi tumor cells at low (1:20) effector-to-target (E:T) ratios in 7 day coculture assays, and produced high levels of IL-2 (669±324 pg/ml), IFN-γ (6715±2541 pg/ml), IL-6 (753±398 pg/ml) and TNF-α (2668±1867 pg/ml). NSG mice engrafted with CD19⁺ EGFPluc-expressing Raji or Daudi tumor cells were treated with variable doses of iC9-CD19.ζ-MC-modified T cells which were capable of eliminating tumor by day 21. However, acute toxicity (>15% body weight reduction) was observed in a CAR-T cell dose dependent manner occuring day 7 to 14 post-CAR-T injection. Systemic administration of 0.5 mg/kg rimiducid rapidly resolved toxicity and mice regained weight within 3-5 days. Serum cytokine concentrations of hIL-6 and hIFN-γ decreased to background levels within 24 hours post-rimiducid treatment. All mice treated with iC9-CD19.ζ-MC-modified T cells demonstrated long-term (>100 days) control of tumor growth and rejected CD19⁺ when rechallenged at day 150. Use of neutralizing antibodies against hIL-6, hIFN-γ and hTNF-α showed that blockade of hTNF-α resulted a significant reduction in weight loss. Further, iC9-CD19.ζ-MC-modified T cells were fractionated into CD4⁺ and CD8⁺ and measured for cytokine production, showing that CD4⁺ CAR-T cells produced higher levels of IL-2 (60-fold), IFN-γ (4-fold), IL-6 (37-fold) and TNF-α (10-fold) compared to CD8⁺ CAR-T cells. Furthermore, treatment of tumor-bearing animals with CD8⁺ selected iC9-CD19.ζ-MC-modified T cells reduced toxicity and the requirement of iC9 activation, while maintaining anti-tumor efficacy.

Conclusions: Enhanced CAR-T cell activity can raise the risk of T cell toxicity. Constitutively active MC costimulation provides robust CAR-T cell proliferation and anti-tumor activity, but also increase cytokine-related toxicity. iC9 can be used to resolve toxicity without affecting long-term efficacy of CAR-T treatment in animals.